JP2020504730A - Ionizable cationic lipids for RNA delivery - Google Patents

Abstract

A compound of formula (I) wherein R1 is a branched alkyl of 10 to 31 carbons and R2 is a linear alkyl, alkenyl or alkynyl of 2 to 20 carbons. Or a branched chain alkyl of 10 to 31 carbons, wherein L1 and L2 are the same or different and are each a linear alkane of 1 to 20 carbons, or a linear alkane of 2 to 20 carbons, respectively. A linear alkene, X 1 is S or O, R 3 is a linear or branched alkylene of 1 to 6 carbons, and R 4 and R 5 are the same or different, Described are compounds of formula (I), or a pharmaceutically acceptable salt thereof, consisting of a compound that is hydrogen or a straight or branched chain alkyl of 1 to 6 carbons.

Description

(Cross-reference of related applications)
This application claims priority to US Patent Application No. 15 / 387,067, filed December 21, 2016.

  Several different types of nucleic acids are currently being developed as therapeutics for the treatment of several diseases. Because these molecules have been developed, they are stable and have a long shelf life to allow encapsulation of nucleic acids without side reactions that may occur in polar aqueous or non-polar solvents. A need has arisen to produce them in a form that can be easily incorporated into anhydrous, organic or anhydrous polar aprotic solvents.

  The description herein relates to novel lipid compositions that facilitate intracellular delivery of biological activity and therapeutic molecules. The description also relates to a pharmaceutical composition comprising such a lipid composition and useful for delivering a therapeutically effective amount of a biologically active molecule into cells of a patient.

  Delivery of a therapeutic compound to a subject is important for its therapeutic effect and can usually be hindered by the compound's limited ability to reach target cells and tissues. Improvement of such compounds to enter target cells of a tissue by various delivery means is important. The description herein relates to novel lipids in compositions and methods of preparation that facilitate targeted intracellular delivery of biologically active molecules.

  Examples of biologically active molecules that often do not achieve effective targeting to patient tissues include immunoglobulin proteins, many proteins, including polynucleotides such as genomic DNA, cDNA, or mRNA antisense polynucleotides; and peptides. Many low molecular weight compounds are mentioned, whether synthetic or naturally occurring, such as hormones and antibiotics.

  One of the fundamental challenges currently faced by healthcare professionals is that several different types of nucleic acids are currently being developed as therapeutics for the treatment of some diseases. These nucleic acids include mRNA for gene expression, DNA, plasmids in gene therapy, low interfering nucleic acids (siNA), siRNA and microRNA (miRNA) for use in RNA interference (RNAi), antisense molecules, ribozymes , Antagomils, and aptamers. As these nucleic acids have been developed, there is a need to produce lipid formulations that are easy to make and can be easily delivered to target tissues.

式中、
Ｒ_１は、１０〜３１個の炭素からなる分枝鎖アルキルであり、
Ｒ_２は、２〜２０個の炭素からなる直鎖アルキル、アルケニル、若しくはアルキニル、又は１０〜３１個の炭素からなる分枝鎖アルキルであり、
Ｌ_１及びＬ_２は、同一であるか又は異なり、それぞれ、１〜２０個の炭素の直鎖アルカン、又は２〜２０個の炭素の直鎖アルケンであり、
Ｘ_１は、Ｓ又はＯであり、
Ｒ_３は、１〜６個の炭素からなる直鎖又は分枝鎖アルキレンであり、
Ｒ_４及びＲ_５は、同一であるか又は異なり、それぞれ、水素又は１〜６個の炭素からなる直鎖若しくは分枝鎖アルキルである化合物からなる、式Ｉの化合物、又は
その薬学的に許容される塩である。 One aspect of what is described is a compound of formula I,

Where:
R ₁ is a branched alkyl of 10 to 31 carbons,
R ₂ is linear alkyl, alkenyl, or alkynyl of 2 to 20 carbons, or branched alkyl of 10 to 31 carbons,
L ₁ and L ₂ are the same or different and are each a straight-chain alkane of 1 to 20 carbons or a straight-chain alkene of 2 to 20 carbons,
X ₁ is S or O;
R ₃ is a linear or branched alkylene of 1 to 6 carbons,
R ₄ and R ₅ are the same or different and each is a compound of formula I comprising a compound which is hydrogen or straight or branched chain alkyl of 1 to 6 carbons, or a pharmaceutically acceptable compound thereof. Is a salt that is

式中、
Ｒ_１は、８、９、１０、１１、１２、１３、１４、１６、１７、１８、１９、２０、２１、又は２２個の炭素の分枝状の非環状アルキル又はアルケニルであり、
Ｌ_１は、１〜１５個の炭素の直鎖アルカンであり、
Ｒ_２は、５、６、７、８、９、１０、１１、１２、１３、１４、若しくは１５個の炭素の直鎖アルキル若しくはアルケニル、又は８、９、１０、１１、１２、１３、１４、１６、１７、１８、１９、２０、２１、若しくは２２個の炭素の分枝状の非環状アルキル若しくはアルケニルであり、
Ｌ_２は、４、５、６、７、８、９、１０、１１、１２、１３、１４、又は１５個の炭素の直鎖アルカンであり、
Ｘは、Ｏ又はＳであり、
Ｒ_３は、１、２、３、４、５、又は６個の炭素の直鎖アルカンであり、
Ｒ_４及びＲ_５は、同一であるか又は異なり、それぞれ、１、２、３、４、５、若しくは６個の炭素の直鎖又は分枝鎖の非環状アルキルである、化合物、
又はその薬学的に許容される塩若しくは溶媒和物である。 Another aspect of what is described is a compound of formula I,

Where:
R ₁ is a branched acyclic alkyl or alkenyl of 8, 9, 10, 11, 12, ₁₃ , 14, 16, 17, 18, 19, 20, 21, or 22 carbons;
L ₁ is a linear alkane of ₁ to 15 carbons,
R ₂ is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons, or 8, 9, 10, 11, 12, 13, 14 , 16, 17, 18, 19, 20, 21, or 22 branched acyclic alkyl or alkenyl of carbon;
L ₂ is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons;
X is O or S;
R ₃ is a linear alkane of 1, 2, ₃ , 4, 5, or 6 carbons;
A compound, wherein R ₄ and R ₅ are the same or different and are each linear or branched non-cyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons;
Or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, R ₁ has 8, 9, 10, 11, 12, 13, 14, 16, or 17 carbons.

In another embodiment, R ₁ comprises two identical alkyl or alkenyl groups.

In another embodiment, R ₁ comprises an alkenyl group.

In another embodiment, R ₂ is alkenyl.

In another embodiment, R ₂ is a branched non-cyclic alkyl.

In another embodiment, _{L 2} is 4,5,6, or having 7 carbons.

Preferably, the compound is of the formula ATX-0082, ATX-0085, ATX-0083, ATX-0121, ATX-0091, ATX-0102, ATX-0098, ATX-0092, ATX-0084, ATX-0095, ATX-0125 , ATX-0094, ATX-0109, ATX-0110, ATX-0118, ATX-0108, ATX-0107, ATX-0093, ATX-0097, and ATX-0096.

式中、
Ｒ_１は、１２、１３、１４、１６、１７、１８、１９、２０、２１、又は２２個の炭素の分枝状の非環状アルキル又はアルケニルであり、
Ｌ_１は、１、２、３、４、５、６、７、８、９、１０、１１、１２、１３、１４、又は１５個の炭素の直鎖アルカンであり、
Ｒ_２は、５、６、７、８、９、１０、１１、１２、１３、１４、若しくは１５個の炭素の直鎖アルキル若しくはアルケニル、又は１０、１１、１２、１３、１４、１６、１７、１８、１９、２０、２１、若しくは２２個の炭素の分枝状の非環状アルキルであり、
Ｌ_２は、１、２、３、４、５、６、７、８、９、１０、１１、１２、１３、１４、又は１５個の炭素の直鎖アルカンであり、
Ｘは、Ｏ又はＳであり、
Ｒ_３は、１、２、３、４、５、又は６個の炭素の直鎖アルカンであり、
Ｒ_４及びＲ_５は、同一であるか又は異なり、それぞれ、１、２、３、４、５、若しくは６個の炭素の直鎖又は分枝鎖の非環状アルキルであり、
化合物の１ｍＭ溶液は、６−（ｐ−トルイジノ）−２−ナフタレンスルホンの蛍光により測定したとき、５．６〜７．０のｐＫａを有し、
化合物は、１０〜１４のｃ−ＬｏｇＤ値を有する、化合物、
又はその薬学的に許容される塩若しくは溶媒和物である。 Another aspect of what is described is a compound of formula I,

Where:
R ₁ is a branched acyclic alkyl or alkenyl of 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons;
L ₁ is a linear alkane of ₁ , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons;
R ₂ is linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons, or 10, 11, 12, 13, 14, 16, 17, , 18, 19, 20, 21, or 22 branched acyclic alkyl of carbon;
L ₂ is a linear alkane of 1, ₂ , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons;
X is O or S;
R ₃ is a linear alkane of 1, 2, ₃ , 4, 5, or 6 carbons;
R ₄ and R ₅ are the same or different and are each linear, branched or non-cyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons;
A 1 mM solution of the compound has a pKa of 5.6-7.0 as measured by 6- (p-toluidino) -2-naphthalene sulfone fluorescence;
The compound having a c-LogD value of 10-14.
Or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, R ₁ has 12, 13, 14, 16, or 17 carbons.

In another embodiment, R ₁ comprises two identical alkyl or alkenyl groups.

In another embodiment, R ₁ comprises an alkyl group.

In another embodiment, R ₂ is alkenyl.

In another embodiment, R ₂ is a branched non-cyclic alkyl.

In another embodiment, L ₁ and L ₂ independently have 1, 2, or 3 carbons.

In another embodiment, both L ₁ and L ₂ have 3 carbons.

In another embodiment, R ₃ is propylene or butylene.

  In another embodiment, the compound has a c-LogD value of at least 11 and its measured pKa is more basic than 6.

In another embodiment, the compound is of the formula ATX-0111, ATX-0132, ATX-0134, ATX-0100, ATX-0117, ATX-0114, ATX-0115, ATX-0101, ATX-0106, ATX-0116, Compounds of ATX-0087, ATX-0058, ATX-0081, ATX-0123, ATX-0122, ATX-0057, ATX-0088, ATX-0087, ATX-0124, ATX-0126, ATX-0129, and ATX-0123 Selected from the group consisting of:

式中、
Ｒ_１は、１２、１３、１４、１６、１７、１８、１９、２０、２１、又は２２個の炭素の分枝状の非環状アルキルであり、
Ｌ_１は、１、２、又は３個の炭素の直鎖アルカンであり、
Ｒ_２は、８、９、１０、１１、若しくは１２の炭素の直鎖アルケニル、又は１２、１３、１４、１６、若しくは１７個の炭素の分枝状の非環状アルキルであり、
Ｌ_２は、１、２、又は３個の炭素の直鎖アルカンであり、
Ｘは、Ｏ又はＳであり、
Ｒ_３は、２又は３個の炭素の直鎖アルカンであり、
Ｒ_４及びＲ_５は、同一であるか又は異なり、それぞれ、１若しくは２個の炭素の直鎖又は分枝鎖の非環状アルキルである、化合物、
又はその薬学的に許容される塩若しくは溶媒和物である。 Another aspect of what is described is a compound of formula I,

Where:
R ₁ is a branched acyclic alkyl of 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons;
L ₁ is a linear alkane of ₁ , 2, or 3 carbons;
R ₂ is linear alkenyl of 8, 9, 10, 11, or 12 carbons, or branched acyclic alkyl of 12, 13, 14, 16, or 17 carbons;
L ₂ is a linear alkane of 1, 2, or 3 carbons;
X is O or S;
R ₃ is a linear alkane of 2 or 3 carbons;
Compounds wherein R ₄ and R ₅ are the same or different and are each linear or branched non-cyclic alkyl of 1 or 2 carbons,
Or a pharmaceutically acceptable salt or solvate thereof.

  In another embodiment, the cationic lipid described herein is present in a pharmaceutical composition. The pharmaceutical composition preferably comprises lipid nanoparticles comprising a nucleic acid, preferably an RNA polynucleotide. Lipid nanoparticles preferably increase the lifespan of circulating RNA. In another embodiment, upon administration of the pharmaceutical composition, the lipid nanoparticles therein deliver the nucleic acid to cells in the body. Preferably, the nucleic acid has an activity to suppress the expression of the target gene. Alternatively, the nucleic acid has the activity of increasing the production of a protein, which encodes when expressed in cells of the body.

  Also described herein is a method for introducing a nucleic acid into mammalian cells by using any of the compositions described above. The cells may be in the liver, lung, kidney, brain, blood, spleen, or bone. The composition is preferably administered intravenously, subcutaneously, intraperitoneally, or intrathecally. Preferably, the compositions described herein are used in a method for treating cancer or an inflammatory disease. The disease can be selected from the group consisting of immune disorders, cancer, kidney disease, fibrotic disease, genetic abnormalities, inflammation, and cardiovascular disease.

1 shows a synthetic route for ATX-0043 from hexanoate (SM1), 4-aminobutanoic acid (SM2), and 4-bromobutyric acid (SM3). Intermediates (Int) 1-8 and the reaction are described in Example 2.

1 shows a synthetic route for ATX-0057 from octanoate (SM1), 4-aminobutanoic acid (SM2), and 4-bromobutyric acid (SM3). Int 1-8 and the reaction are described in Example 3.

FIG. 3 shows the same synthetic route of ATX-0058 from SM1, SM2 and SM3 as FIG. Int 1-7 and the reaction are described in Example 4.

FIG. 3 shows the same ATX-0081 synthesis route from SM1, SM2 and SM3 as in FIG. Int 1-8 and the reaction are described in Example 5.

FIG. 3 shows a synthesis route of ATX-0082 from SM1, SM2, and SM3, which is the same as FIG. Int 1-7 and the reaction are described in Example 6.

FIG. 3 shows the same synthetic route of ATX-0086 from SM1, SM2 and SM3 as in FIG. Int 1-8 and the reaction are described in Example 7.

FIG. 3 shows the same synthetic route of ATX-0087 from SM1, SM2, and SM3 as FIG. Int 1-8 and the reaction are described in Example 8.

FIG. 3 shows the same synthetic route of ATX-0088 from SM1, SM2 and SM3 as in FIG. Int 1-8 and the reaction are described in Example 9.

FIG. 3 shows the same synthetic route of ATX-0083 from SM1, SM2, and SM3 as FIG. Int 1-8 and the reaction are described in Example 10.

FIG. 3 shows the same synthetic route of ATX-0084 from SM1, SM2, and SM3 as FIG. Int 1-8 and the reaction are described in Example 11.

2 shows a synthesis route of ATX-0061 from SM1 and SM2, which is the same as FIG. Int 1-5 and the reaction are described in Example 12.

FIG. 2 shows a synthesis route of ATX-0063 from SM1 and SM2, which is the same as FIG. Int 1-5 and the reaction are described in Example 13.

FIG. 2 shows a synthesis route of ATX-0064 from SM1 and SM2, which is the same as FIG. Int 1-5 and the reaction are described in Example 14.

1 shows a synthesis route of ATX-0081. Int 1-6 and the reaction are described in Example 15.

FIG. 3 shows the same ATX-0085 synthesis route from SM1, SM2, and SM3 as in FIG. Int 1-11 and the reaction are described in Example 16.

1 shows the synthesis route of ATX-0134. Int 1-6 and the reaction are described in Example 17.

0.03 mg / kg in a nanoparticle comprising ATX-002, ATX-0057, ATX-0081, ATX-0082, ATX-0083, ATX-0084, ATX-0085, ATX-0087, or ATX-0087 cationic lipid and 9 shows EPO mRNA levels (ng / mL) after injection of 0.1 mg / kg mRNA into mice.

9 shows the anti-factor VII knockdown activity of ATX-0057 and liposomes having ATX-0058 versus the activity of ATX-002 and control (PBS alone).

Figure 5 shows the anti-EPO knockdown activity of liposomes with ATX-0057 versus the activity of ATX-002.

Definitions "At least one" means one or more (e.g., 1-3, 1-2, or 1).

  "Composition" means a product that contains a specified component in a specified amount, as well as any product that results, directly or indirectly, from a specified amount of a combination of the specified components.

  "In combination with" refers to the administration of a compound of Formula I with another agent in a method of treatment of the invention, wherein the compound of Formula I and the other agent are administered sequentially or simultaneously in separate dosage forms. Or administered simultaneously in the same dosage form.

  "Mammal" means a human or other mammal, or a human.

  "Patient" means both human and other mammals, preferably humans.

"Alkyl" means a saturated or unsaturated, straight or branched hydrocarbon chain. In various embodiments, the alkyl group has 1 to 18 carbons, i.e. whether it is _C 1 to C1 ₈ group, or _C 1 _{-C 12 group,} C 1 -C ₆ group, or _C 1 ~ C ₄ groups. Independently, in various embodiments, an alkyl group has zero (ie, straight) branches, one branch, two branches, or three or more branches. “Alkenyl” is an unsaturated alkyl that can have one double bond, two double bonds, or more than two double bonds. “Alkynyl” is an unsaturated alkyl that can have one triple bond, two triple bonds, or more than two triple bonds. The alkyl chain is optionally substituted with one substituent (i.e., the alkyl group is mono-substituted), or 1-2 substituents, or 1-3 substituents, or 1-4 substituents, and the like. obtain. Substituents can be selected from the group consisting of hydroxy, amino, alkylamino, boronyl, carboxy, nitro, cyano, and the like. If an alkyl group incorporates one or more heteroatoms, the alkyl group is referred to herein as a heteroalkyl group. If the substituent on the alkyl group is a hydrocarbon, the resulting group is simply called a substituted alkyl. In various aspects, the alkyl group containing a substituent is 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or It has less than 7 carbons.

  "Lower alkyl" means a group having 1-6 carbons in the chain which may be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and hexyl.

  "Alkoxy" means an alkyl-O- group in which alkyl is as previously defined. Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and heptoxy. The bond to the parent moiety is through the ether oxygen.

  "Alkoxyalkyl" means an alkoxy-alkyl- group in which the alkoxy and alkyl are as previously described. Preferred alkoxyalkyls contain a lower alkyl group. The bond to the parent moiety is through the alkyl.

  "Alkylaryl" means an alkyl-aryl- group in which the alkyl and aryl are as previously described. Preferred alkylaryls contain a lower alkyl group. The bond to the parent moiety is through the aryl.

  "Aminoalkyl" means an NH2-alkyl- group in which the alkyl is as defined above, attached to the parent moiety through an alkyl group.

  "Carboxyalkyl" means a HOOC-alkyl- group in which the alkyl is as defined above, attached to the parent moiety through the alkyl group.

  "Commercially available chemicals" and the chemicals used in the examples described herein can be obtained from standard commercial sources, such as, for example, Acros Organics ( Pittsburgh, Pa.), Sigma-Adrich Chemical (Milwaukee, WIS.), Avocado Research (Lancashire, UK), Bionet (Cornwall, UK), Boron Morgane. Combi-Blocks (San Diego, Calif.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY). , Fisher Scientific Co .; (Pittsburgh, Pa.), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. (Cornwall, UK), Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland, Oreg.), And Wako Chemicals USA, Inc. (Richmond, Va.).

  "Compounds described in the chemical literature" can be identified through reference books and databases directed to chemical compounds and chemical reactions, as known to those skilled in the art. Suitable references and articles which detail the synthesis of reactants useful in the preparation of the compounds disclosed herein, or provide references to literature describing the preparation of the compounds disclosed herein. Include, for example, "Synthetic Organic Chemistry", John Wiley and Sons, Inc. New York, S.M. R. Sandler et al, "Organic Functional Group Preparations," 2nd Ed. , Academic Press, New York, 1983; O. House, "Modern Synthetic Reactions," 2nd Ed. , W.S. A. Benjamin, Inc. Menlo Park, Calif. , 1972; L. Glichrist, "Heterocyclic Chemistry," 2nd Ed. John Wiley and Sons, New York, 1992; March, "Advanced Organic Chemistry: reactions, Mechanisms and Structure," 5th Ed. , Wiley Interscience, New York, 2001; certain and similar reactants are also prepared by the Chemical Abstract Service of the American Chemical Society, a chemical known to the public that is available in most public and university libraries. And may be identified via an online database (you can contact the American Chemical Society, Washington, DC for details). Chemicals known in the catalog but not commercially available can be prepared by custom chemical synthesis houses, and many of the standard chemical supply houses (such as those listed above) provide custom synthesis services. ing.

  "Halo" means a fluoro, chloro, bromo, or iodo group. Fluoro, chloro, or bromo are preferred, and fluoro and chloro are more preferred.

  "Halogen" means fluorine, chlorine, bromine, or iodine. Fluorine, chlorine and bromine are preferred.

  "Heteroalkyl" means saturated or unsaturated, straight or branched, a chain containing carbon, and at least one heteroatom. A heteroalkyl group can, in various embodiments, have one heteroatom, or 1-2 heteroatoms, or 1-3 heteroatoms, or 1-4 heteroatoms. In one aspect, the heteroalkyl chain contains 1-18 (ie, 1-18) member atoms (carbon and heteroatoms), and in various embodiments, 1-12, or 1-6, Or contains 1 to 4 member atoms. Independently, in various embodiments, a heteroalkyl group has zero branches (ie, is linear), one branch, two branches, or three or more branches. Independently, in one embodiment, the heteroalkyl group is saturated. In another embodiment, a heteroalkyl group is unsaturated. In various embodiments, the unsaturated heteroalkyl is one double bond, two double bonds, three or more double bonds, and / or one triple bond, two triple bonds, or three or more triple bonds. It may have a bond. Heteroalkyl chains can be substituted or unsubstituted. In one embodiment, the heteroalkyl chain is unsubstituted. In another embodiment, the heteroalkyl chain is substituted. A substituted heteroalkyl chain can have one substituent (ie, by monosubstitution), or have, for example, one to two substituents, or one to three substituents, or one to four substituents. I can do it. Exemplary heteroalkyl substituents include esters (-C (O) -OR) and carbonyl (-C (O)-).

  "Hydroxyalkyl" means a HO-alkyl group in which alkyl is as previously defined. Preferred hydroxyalkyls contain a lower alkyl. Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

"Hydrate" is a solvent molecule means a solvate wherein H _{2 O.}

  "Lipid" means an organic compound that contains an ester of a fatty acid and is characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually of at least three classes: (1) "simple lipids" including fats and oils and waxes, (2) phospholipids, glycolipids, cationic lipids, non-cationic lipids, neutral lipids, and anionics “Complex lipids” including lipids, all described in more detail herein, as well as (3) classified as “derived lipids” such as steroids.

  “Lipid particles” refers to lipid formulations that can be used to deliver a therapeutic nucleic acid (eg, mRNA) to a target site of interest (eg, cells, tissues, organs, etc.). In preferred embodiments, the lipid particles are typically formed from cationic lipids, non-cationic lipids (eg, phospholipids), complex lipids that prevent aggregation of the particles (eg, PEG lipids), and optionally cholesterol. Nucleic acid-lipid particles. Typically, the therapeutic nucleic acid (eg, mRNA) can be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

  The lipid particles are typically 30-150 nm, 40-150 nm, 50-150 nm, 60-130 nm, 70-110 nm, 70-100 nm, 80-100 nm, 90-100 nm, 70-90 nm, 80-90 nm, 70 nm. -80 nm, or 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, It has an average diameter of 145 nm or 150 nm and is substantially non-toxic. In addition, nucleic acids, when present in the lipid particles of the invention, are resistant in aqueous solution to degradation by nucleases.

  "Lipid-encapsulated" means lipid particles that provide a therapeutic nucleic acid, such as mRNA, either fully encapsulated, partially encapsulated, or both. In a preferred embodiment, the nucleic acid (eg, mRNA) is completely encapsulated in the lipid particles.

  “Lipid complex” means a complex lipid that inhibits aggregation of lipid particles. Such lipid conjugates include PEG-lipid conjugates, such as PEG conjugated to dialkyloxypropyl (eg, PEG-DAA conjugate), PEG conjugated to diacylglycerol (eg, PEG-DAG conjugate) ), PEG conjugated to cholesterol, PEG conjugated to phosphatidylethanolamine, and PEG conjugated to ceramide, cationic PEG lipids, polyoxazoline (POZ) -lipid conjugates, polyamide oligomers, and mixtures thereof. But not limited to these. PEG or POZ can be conjugated directly to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for attaching PEG or POZ to a lipid can be used, including non-ester containing linker moieties and ester containing linker moieties. In certain preferred embodiments, non-ester containing linker moieties such as amides or carbamates are used.

  "Amphiphilic lipid" means a material in which the hydrophobic portion of the lipid material is oriented into the hydrophobic phase while the hydrophilic portion is oriented toward the aqueous phase. The hydrophilic character comes from the presence of polar or charged groups such as carbohydrates, phosphoric acids, carboxylic acids, sulfatos, aminos, sulfhydryls, nitros, hydroxyls and other similar groups. Hydrophobicity includes long chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group (s). , Including but not limited to, non-polar groups. Examples of amphiphilic compounds include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

  Representative examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dioleoylphosphatidylcholine, distearoyl thiocholine. , And dilinoleoylphosphatidylcholine, but are not limited thereto. Other phosphorus-free compounds such as sphingolipids, glycosphingolipid family, diacylglycerols, and β-acyloxy acids are also within the group designated as amphiphilic lipids. In addition, the above amphiphilic lipids can be mixed with other lipids including triglycerides and sterols.

  "Neutral lipid" refers to a lipid species that exists at a selected pH in either the uncharged or neutral zwitterionic form. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

  "Non-cationic lipid" means an amphipathic or neutral or anionic lipid and is described in more detail below.

  "Anionic lipid" means a lipid that is negatively charged at physiological pH. Examples of these lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidylic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyl oleyl phosphatidyl. Examples include, but are not limited to, glycerol (POPG), and other anionic modifying groups conjugated to neutral lipids.

  “Hydrophobic lipids” are those long-chain saturated and unsaturated aliphatic hydrocarbon groups, and those optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group (s). A compound having a nonpolar group, including, but not limited to, a group. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane. Not done.

“Cationic lipid” and “aminolipid” are used interchangeably and refer to one, two, three or more fatty acid or aliphatic alkyl chains and a pH titratable amino head group (eg, alkylamino or dialkylamino). Head group) and salts thereof. Cationic lipids are typically protonated at a pH below _the pK _a of cationic lipid (have ie positively charged), which is substantially neutral in pH above pK _a. The cationic lipids of the present invention may also be referred to as titratable cationic lipids. In some embodiments, the cationic lipid is a tertiary amine (eg, pH titratable) head group that can be protonated, wherein each alkyl chain is independently 0-3 (eg, 0, 1, 2,. Or 3) a _C18 alkyl chain having a double bond, and an ether, ester, or ketal bond between the head group and the alkyl chain. Such cationic lipids are also known as DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (DLin-C2K-DMA, XTC2, and C2K) ), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin- M-C3-DMA (also known as MC3), and (DLin-MP-DMA) (also known as 1-B11), but are not limited to these.

  "Substituted" refers to substitution with a particular group other than hydrogen, or with one or more groups, moieties, or radicals that may be the same or different, each being, for example, independently selected.

  An “antisense nucleic acid” is a non-sense nucleic acid that binds to a target RNA through RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365,566) interaction and changes the activity of the target RNA. By enzymatic nucleic acid molecule (for review, see Stein and Cheng, 1993 Science 261, 1004 and U.S. Patent No. 5,849,902 to Woolf et al.). Typically, an antisense molecule is complementary to a target sequence along a single contiguous sequence of the antisense molecule. However, in certain embodiments, the antisense molecule can bind to the substrate such that the substrate molecule forms a loop, and / or the antisense molecule binds such that the antisense molecule forms a loop. can do. Thus, an antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences, or two (or even more) non-contiguous The sequence portion may be complementary to the target sequence, or both. In addition, antisense DNA can be used to target RNA by DNA-RNA interaction, thereby activating RNase H, which digests the target RNA in duplex. Antisense oligonucleotides can include one or more RNAse H activation regions that can activate RNAse H cleavage of the target RNA. Antisense DNA can be chemically synthesized or expressed by use of a single-stranded DNA expression vector or its equivalent. “Antisense RNA” is an RNA strand having a sequence complementary to a target gene mRNA, which can induce RNAi by binding to the target gene mRNA. “Antisense RNA” is an RNA strand having a sequence complementary to a target gene mRNA, and is considered to induce RNAi by binding to the target gene mRNA. "Sense RNA" has a sequence complementary to the antisense RNA and anneals to the complementary antisense RNA to form iNA. These antisense and sense RNAs have been conventionally synthesized with an RNA synthesizer.

  "Nucleic acid" means deoxyribonucleotides or ribonucleotides in single or double stranded form and polymers thereof. The term is a known nucleotide analog or modified backbone residue that has similar binding properties as a reference nucleic acid and is metabolized, synthesized, naturally occurring, and non-naturally occurring in a manner similar to the reference nucleotide. Or a nucleic acid containing a linkage. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNA).

  "RNA" means a molecule that contains at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide having a hydroxyl group at the 2 'position of the [beta] -D-ribo-furanose moiety. The term refers to isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and Modified RNA different from RNA naturally occurring by the addition, deletion, substitution, and / or modification of the above nucleotides is included. Such modifications may include adding non-nucleotide material, such as at the end (s) of the interfering RNA, or internally, for example, at one or more nucleotides of the RNA. The nucleotides in the RNA molecules of the invention can also include non-naturally occurring nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA. As used herein, the terms “ribonucleic acid” and “RNA” refer to at least one ribonucleotide, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, non-coding RNA, and multivalent RNA. Refers to a molecule containing a residue. "Ribonucleotides" are nucleotides having a hydroxyl group at the 2'-position of the [beta] -D-ribo-furanose moiety. These terms include isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and Includes modified and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, modification, and / or modification of one or more nucleotides. Modification of the RNA may include adding non-nucleotide material, such as to the end (s) of the interfering RNA, or internally, for example, at one or more of the nucleotides of the RNA nucleotides in the RNA molecule. Or non-standard nucleotides such as chemically synthesized nucleotides or deoxylinucleotides. These modified RNAs may be referred to as analogs.

  "Nucleotide" refers to natural bases (standard) and modified bases well known in the art. Such bases are generally located at the 1 'position of the nucleotide sugar moiety. Nucleotides generally contain a base, a sugar, and a phosphate group. Nucleotides may be unmodified or modified at the sugar, phosphate, and / or base moieties (also referred to as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, and the like; See Usman and McSwiggen, Eckstein et al., WO 92/07065, Usman et al., WO 93/15187, supra, Uhlman & Peyman, all incorporated herein by reference). Limbach, et al, Nucleic Acids Res. 22: 2183, 1994, there are several examples of modified nucleobases known in the art. Some non-limiting examples of base modifications that can be introduced into nucleic acid molecules include inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-tri Methoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (eg, 5-methylcytidine), 5-alkyluridine (eg, ribothymidine), 5-halouridine (eg, 5-bromouridine) Or 6-azapyrimidine or 6-alkylpyrimidine (for example, 6-methyluridine), propyne and the like (Burgin, et al., Biochemistry 35: 14090, 1996; Uhlman & Peyman, supra). The “modified base” in this embodiment means a nucleotide base other than adenine, guanine, cytosine, and uracil at the 1′-position, or an equivalent thereof.

  "Complementary nucleotide bases" refers to pairs of nucleotide bases that form hydrogen bonds with one another. Adenine (A) pairs with thymine (T) or uracil (U) in the RNA, and guanine (G) pairs with cytosine (C). Complementary segments or strands of nucleic acids that hybridize to each other (ie, join by hydrogen bonding). By "complementary" is meant that the nucleic acid can form hydrogen bond (s) with another nucleic acid sequence, either by traditional Watson-Crick or other non-traditional binding modes. .

  "MicroRNA" (miRNA) refers to a single-stranded RNA molecule of 21-23 nucleotides in length that regulates gene expression, where miRNA is encoded by a gene transcribed from DNA but not translated into protein (non-coding RNA). Instead, they are processed from primary transcripts, known as pri-miRNAs, into short stem-loop structures called pre-miRNAs, and finally into functional miRNAs. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to down-regulate gene expression.

  “Low interfering RNA (siRNA)” and “short interfering RNA” and “silencing RNA” refer to a class of double-stranded RNA molecules that play a variety of biological functions and are 16-40 nucleotides in length. . Most notably, siRNAs participate in the RNA interference (RNAi) pathway, which interferes with the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also function in RNAi-related pathways, such as as antiviral mechanisms or in forming genomic chromatin structures, and the complexity of these pathways is now being elucidated. is there.

  “RNAi” is RNA-dependent initiated by short double-stranded RNA molecules in the cell that is regulated by the RNA-induced silencing complex (RISC), which interacts with the catalytic RISC component argonaute Refers to the sex gene silencing process. If the double-stranded RNA or RNA-like iNA or siRNA is exogenous (resulting from infection by a virus having an RNA genome or from transfected iNA or siRNA), the RNA or iNA is transferred directly into the cytoplasm and the enzyme Dicer cuts into short pieces. The starting dsRNA may also be endogenous (derived from a cell), such as a pre-microRNA expressed from an RNA-encoding gene in the genome. Primary transcripts from such genes are first processed to form the characteristic stem-loop structure of the pre-miRNA in the nucleus and then translocated to the cytoplasm, which is cleaved by Dicer. Thus, the two exogenous and endogenous dsRNA pathways converge in the RISC complex. The active component of the RNA-induced silencing complex (RISC) is an endonuclease called Argonaute protein that cleaves the target mRNA strand complementary to their bound siRNA or iNA. Since the fragments produced by Dicer are double-stranded, they can theoretically produce functional siRNA or iNA, respectively. However, only one of the two chains, known as the guide strand, binds to Argonaute protein and directs gene silencing. Other anti-guide or passenger strands are degraded during RISC activation.

  Compounds of formula I

  Reference to a compound of Formula I herein is understood to include reference to salts thereof, unless otherwise indicated. As used herein, the term "salt (s)" refers to acidic salts formed with inorganic and / or organic acids, and basic salts formed with inorganic and / or organic bases. In addition, if a compound of Formula I contains both a basic moiety, such as, but not limited to, pyridine or imidazole and an acidic moiety, such as, but not limited to, a carboxylic acid, a zwitterion ("inner salt") is formed. And is included within the term "salt (s)" as used herein. The salt can be a pharmaceutically acceptable (ie, non-toxic, physiologically acceptable) salt, but other salts are also useful. A salt of a compound of formula I can be obtained, for example, by reacting the compound of formula I with an amount of an acid or base, such as an equivalent, in a medium such as that in which the salt precipitates or in an aqueous medium, followed by lyophilization Can be formed by

  Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate , Camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanate, glycerophosphate, hemisulfate, heptanoate, Hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, Nitrate, oxalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, Haq, sulfate, sulfonates (such as those mentioned herein), tartrates, thiocyanates, (also known as tosylates) toluenesulfonate undecanoates, and the like. In addition, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are described, for example, in Berge et al, J. Mol. Pharmaceutical Sciences (1977) 66 (1) 1-19; Gould, International J.C. Pharmaceutics (1986) 33 201-217, Anderson et al. , The Practice of Medicinal Chemistry (1996), Academic Press, New York, and The Orange Book (Food & Drug Administration, Washington, D.C.). These disclosures are incorporated herein by reference.

  Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (eg, organic amines), for example, , Benzathine, dicyclohexylamine, hydravamin (formed with N, N-bis (dehydroabiethyl) ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, arginine or lysine, etc. And salts with amino acids. Basic nitrogen-containing groups include lower alkyl halides (eg, methyl, ethyl, propyl, and butyl chloride, bromide, and iodide), dialkyl sulfates (eg, dimethyl, diethyl, dibutyl, and diamyl sulfate), long chain They can be quaternized with agents such as halides (eg, decyl, bromide, and decyl, lauryl, myristyl, and stearyl iodides), arylalkyl halides (eg, benzyl bromide and phenethyl).

  All such acid and base salts are intended to be pharmaceutically acceptable salts within the scope of the present disclosure, and all acid and base salts are, for the purposes of this disclosure, the corresponding compounds of formula I Is considered to be equivalent to the free form of the compound.

  The compounds of Formula I can exist in unsolvated and solvated forms, including hydrated forms. In general, solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc., are equivalent to unsolvated forms for the purposes of this disclosure.

  The compounds of formula I and salts, solvates thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present disclosure.

  Also, polymorphs of the compounds of the present disclosure are within the scope of the present disclosure (ie, polymorphs of the compound of formula I are within the scope of the present disclosure).

  All stereoisomers (eg, geometric isomers, optical isomers, etc.) of the present compounds (including salts, solvates, and prodrugs of the compounds, and salts and solvates of the prodrugs), for example, What can be present with an asymmetric carbon on various substituents, including enantiomeric forms (which can also exist in the absence of an asymmetric carbon), rotamer forms, atropisomers, and diastereomeric forms are disclosed in the present disclosure. Within the scope of An individual stereoisomer of a compound of the disclosure may, for example, be substantially free of other isomers, or, for example, as a racemate, or all other or other selected stereoisomers. It may be mixed with the body. The chiral centers of the compounds herein may have the S or R configuration as defined by IUPAC 1974 Recommendations. Use of the terms "salt", "solvate" and the like applies equally to salts and solvates of the enantiomers, stereoisomers, rotamers, tautomers, racemates, or prodrugs of the disclosed compounds. It is intended to.

Classes of compounds that can be used as chemotherapeutic agents (anti-tumor agents) include alkylating agents, antimetabolites, natural products and their derivatives, hormones and steroids (including synthetic analogs), and synthetic drugs. Is mentioned. Examples of compounds within these classes are shown below.
Lipid particles

  The compounds of formula I include their pharmaceutically acceptable salts in a lipid composition comprising nanoparticles or bilayers of lipid molecules. The lipid bilayer preferably further comprises a neutral lipid or polymer. The lipid composition preferably comprises a liquid medium. The composition preferably further encapsulates the nucleic acid. The nucleic acid preferably has an activity of suppressing the expression of the target gene by utilizing RNA interference (RNAi). The lipid composition preferably further comprises a nucleic acid and a neutral lipid or polymer. The lipid composition preferably encapsulates the nucleic acid.

  The description provides a lipid particle comprising one or more therapeutic mRNA molecules encapsulated within the lipid particle.

  In some embodiments, the mRNA is completely encapsulated within the lipid portion of the lipid particle such that the mRNA in the lipid particle is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid particles described herein are substantially non-toxic to mammals, such as humans. Lipid particles typically have an average diameter of 30 nm to 150 nm, 40 nm to 150 nm, 50 nm to 150 nm, 60 nm to 130 nm, 70 nm to 110 nm, or 70 to 90 nm. The lipid particles of the invention are typically 1: 1 to 100: 1, 1: 1 to 50: 1, 2: 1 to 25: 1, 3: 1 to 20: 1, 5: 1 to 15: It also has a lipid: RNA ratio (mass / mass ratio) of 1, or 5: 1 to 10: 1, or 10: 1 to 14: 1, or 9: 1 to 20: 1. In one embodiment, the lipid particles have a lipid: RNA ratio (mass / mass ratio) of 12: 1. In another embodiment, the lipid particles have a lipid: mRNA ratio (mass / mass ratio) of 13: 1.

  In a preferred embodiment, the lipid particles comprise mRNA, cationic lipids (eg, one or more cationic lipids or salts thereof as described herein), phospholipids, and complex lipids that inhibit aggregation of the particles (eg, One or more PEG lipid conjugates). The lipid particles may also include cholesterol. The lipid particles can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mRNAs that express one or more polypeptides.

  In nucleic acid-lipid particles, mRNA can be completely encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In a preferred embodiment, the lipid particles comprising the mRNA are completely encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In certain cases, the mRNA in the lipid particles does not substantially degrade after exposure of the particles to nuclease at 37 ° C. for at least 20, 30, 45, or 60 minutes. In certain other cases, the mRNA in the lipid particles is at least 30, 45, or 60 minutes at 37 ° C, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 14 , 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours without substantial degradation after incubation of the particles in serum. In other embodiments, the mRNA is conjugated to the lipid portion of the particle. One of the advantages of the formulations of the present invention is that the nucleic acid-lipid particle composition is substantially non-toxic to mammals, such as humans.

  "Fully encapsulated" means that the nucleic acids (e.g., mRNA) in the nucleic acid-lipid particles are not significantly degraded after exposure to a serum or nuclease assay that will significantly degrade free RNA. . When fully encapsulated, preferably less than 25% of the nucleic acids in the particle are degraded in a process that will usually degrade 100% of free nucleic acid, more preferably less than 10% of the particle, most preferably Less than 5% of the nucleic acids are degraded. "Completely encapsulated" also means that upon in vivo administration, the nucleic acid-lipid particles do not rapidly degrade into their components.

In the context of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with a nucleic acid. Encapsulation is determined by adding the dye to the liposome formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of a non-ionic detergent. Detergent-mediated disruption of the liposome bilayer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid _{encapsulated, E = (I 0 -I)} / I can be calculated as _0, wherein, / and _{I 0} refers to the fluorescence intensity before and after the addition the addition of the detergent.

  In another embodiment, the present invention provides a nucleic acid-lipid particle composition comprising a plurality of nucleic acid-lipid particles.

  Lipid particles comprise mRNA completely encapsulated within the lipid portion of the particle, such that 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%. %, 80% to 100%, 90% to 100%, 30% to 95%, 40% to 95%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 85% to 95%, 90% to 95%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, or at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, or 99% (or any percentage or range thereof) of particles Having encapsulated mRNA in.

Depending on the intended use of the lipid particles, the ratio of the components can be varied, and the efficiency of delivery of a particular formulation can be measured using assays known in the art.
Cationic lipid

  The description includes the synthesis of certain cationic lipid compounds. The compounds are particularly suitable for delivering polynucleotides to cells and tissues, as shown in the following sections. The lipomacrocycle compounds described herein may be used for other purposes, and for example, for recipients and additives.

  The cationic lipid compound can be synthesized using techniques in the art. One skilled in the art will recognize other methods for making these compounds and for making other compounds of the description.

  Cationic lipid compounds may be combined with an agent to form microparticles, nanoparticles, liposomes, or micelles. The drug delivered by the particle, liposome, or micelle can be in gaseous, liquid, or solid form, and the drug can be a polynucleotide, protein, peptide, or small molecule. The lipomacrocycle compound may form particles in combination with other cationic lipid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, or lipids. These particles may then be optionally combined with a pharmaceutical excipient to form a pharmaceutical composition.

  The present description provides novel cationic lipid compounds and drug delivery systems based on the use of such cationic lipid compounds. The system can be used in pharmaceutical / drug delivery technology to deliver a polynucleotide, protein, small molecule, peptide, antigen, or drug to a patient, tissue, organ, or cell. These novel compounds can also be used as coatings, additives, excipients, materials, or materials for biotechnology.

  The cationic lipid compounds of the present description offer several different uses in drug delivery technology. The amine-containing portion of the cationic lipid compound can be used to conjugate polynucleotides, thereby enhancing delivery of the polynucleotides and preventing their degradation. Cationic lipid compounds can also be used in the formation of picoparticles, nanoparticles, microparticles, liposomes, and micelles containing the agent to be delivered. Preferably, the cationic lipid compound is biocompatible and biodegradable, and the particles formed are also biodegradable and biocompatible, and are used to provide controlled, sustained release of the delivered agent. obtain. These and their corresponding particles can also respond to pH changes, given that they are protonated at lower pH. They can also function as proton sponges in the delivery of drugs to cells, causing endosomal lysis.

In certain embodiments, the cationic lipid compound is relatively non-toxic. Cationic lipid compounds can be biocompatible and biodegradable. Cationic lipids may have about 5.5 to about 7.5, more preferably in the range of from about 6.0 to about 7.0 measured pK _a (in the formulation environment). It can be designed to have _a desired pKa of about 3.0 to about 9.0 or about 5.0 to about 8.0. The cationic lipid compounds described herein are particularly attractive for drug delivery for several reasons: they interact with DNA, RNA, other polynucleotides, and other negatively charged drugs Therefore, they contain amino groups to protect the delivered drug, to buffer the pH, for endo-osmolysis, and can be synthesized from commercially available starting materials, and / or or they are pH responsive and can be operated at a desired pK _a.
Neutral helper lipid

Non-limiting examples of non-cationic lipids include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside. , Dicetyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DPPG) DOPE), palmitoyl oleoyl-phosphatidylcholine (POP ), Palmitoyl oleoyl-phosphatidylethanolamine (POPE), palmitoyl oleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), Dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielideyl-phosphatidylethanolamine (DEPE) , Stearoyl oleoyl-phosphatidylethanolamine (SOPE), lyso Scan choline, dilinoleoyl phosphatidylcholine, and include phospholipids, such as mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably fatty acids having C ₁₀ -C ₂₄ carbon chains, for example, an acyl group derived from lauroyl, myristoyl, palmitoyl, stearoyl, or oleyl.

  Further examples of non-cationic lipids include sterols such as cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include 5α-cholestanol, 5α-coprostanol, cholesteryl- (2′-hydroxy) -ethyl ether, cholesteryl- (4′-hydroxy) -butyl ether, and 6-ketocholestanol Polar analogs such as 5α-cholestane, cholesterone, 5α-cholestanone, 5α-cholestanone, and cholesteryl decanoate; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl- (4'-hydroxy) -butyl ether.

  In some embodiments, the non-cationic lipid present in the lipid particles comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the non-cationic lipid present in the lipid particles comprises or consists of one or more phospholipids, for example, a cholesterol-free lipid particle formulation. In yet other embodiments, the non-cationic lipid present in the lipid particles comprises or consists of a cholesterol or derivative thereof, eg, a phospholipid-free lipid particle formulation.

  Other examples of non-cationic lipids include, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, Phosphorus-free containing lipids such as alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

  In some embodiments, the non-cationic lipid comprises 10 mol% to 60 mol%, 20 mol% to 55 mol%, 20 mol% to 45 mol%, 20 mol% to 40 mol%, 25 mol% to 50 mol% of the total lipid present in the particles. , 25 mol% to 45 mol%, 30 mol% to 50 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 35 mol% to 45 mol%, 37 mol% to 42 mol%, or 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, or 45 mol% (or any ratio or range thereof).

  In embodiments where the lipid particles contain a mixture of phospholipids and cholesterol or a cholesterol derivative, the mixture may contain up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipids present in the particles. .

  In some embodiments, the phospholipid component in the mixture comprises 2 mol% to 20 mol%, 2 mol% to 15 mol%, 2 mol% to 12 mol%, 4 mol% to 15 mol%, or 4 mol% of total lipids present in the particles. % To 10 mol% (or any ratio or range thereof). In certain preferred embodiments, the phospholipid component in the mixture comprises 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 6 mol% of the total lipids present in the particles. % To 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any ratio or range thereof).

  In other embodiments, the cholesterol component in the mixture comprises 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 27 mol% to 37 mol% of total lipids present in the particles. %, 25 mol% to 30 mol%, or 35 mol% to 40 mol% (or any proportion or range thereof). In certain preferred embodiments, the cholesterol component in the mixture comprises 25 mol% to 35 mol%, 27 mol% to 35 mol%, 29 mol% to 35 mol%, 30 mol% to 35 mol%, 30 mol% of the total lipids present in the particles. -34 mol%, 31 mol%-33 mol%, or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, or 35 mol% (or any ratio or range thereof).

  In embodiments where the lipid particles do not comprise a phospholipid, cholesterol or a derivative thereof comprises up to 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipids present in the particles. %.

  In some embodiments, cholesterol or a derivative thereof in the phospholipid-free lipid particle formulation comprises 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% of total lipids present in the particles. -40 mol%, 31 mol%-39 mol%, 32 mol%-38 mol%, 33 mol%-37 mol%, 35 mol%-45 mol%, 30 mol%-35 mol%, 35 mol%-40 mol%, or 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, or 40 mol% (or any proportion or range thereof).

  In other embodiments, the non-cationic lipid comprises 5 mol% to 90 mol%, 10 mol% to 85 mol%, 20 mol% to 80 mol%, 10 mol% (eg, phospholipid only), or 60 mol% of the total lipid present in the particles. % (Eg, phospholipids and cholesterol or derivatives thereof) (or any percentage or range thereof).

  The percentage of non-cationic lipid present in the lipid particles is the target amount, and the actual amount of non-cationic lipid present in the formulation can vary, for example, ± 5 mol%.

  A composition containing a cationic lipid compound can be 30-70% cationic lipid compound, 0-60% cholesterol, 0-30% phospholipid, and 1-10% polyethylene glycol (PEG). Preferably, the composition is 30-40% cationic lipid compound, 40-50% cholesterol, and 10-20% PEG. In another preferred embodiment, the composition is 50-75% cationic lipid compound, 20-40% cholesterol, and 5-10% phospholipid, and 1-10% PEG. The composition may contain 60-70% cationic lipid compound, 25-35% cholesterol, and 5-10% PEG. The composition may contain up to 90% of a cationic lipid compound and 2-15% of a helper lipid.

Formulations include, for example, 8-30% compound, 5-30% helper lipid, and 0-20% cholesterol; 4-25% cationic lipid, 4-25% helper lipid, 2-25% Cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30% cationic lipids, 2-30% helper lipids, 1-15% cholesterol, 2-35% cholesterol -PEG and 1-20% cholesterol-amines; or lipid particle formulations containing up to 90% cationic lipids and 2-10% helper lipids, or even 100% cationic lipids.
Lipid complex

  In addition to cationic, the lipid particles described herein may further include a lipid complex. Complex lipids are useful in preventing aggregation of the particles. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.

  In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examples of PEG-lipids include PEG conjugated to dialkyloxypropyl (PEG-DAA), PEG conjugated to diacylglycerol (PEG-DAG), PEG conjugated to phospholipids such as phosphatidylethanolamine (PEG-PEG-DAG). PE), PEG conjugated to ceramide, PEG conjugated to cholesterol or its derivatives, and mixtures thereof.

PEG is a linear, water-soluble polymer of ethylene PEG repeating units having two terminal hydroxyl groups. PEGs are classified by their molecular weight and include: monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimidyl succinate (MePEG-OH). S-NHS), monomethoxypolyethylene glycol - amine _(MePEG-NH 2), mono-methoxy polyethylene glycol - tresylate (MePEG-TRES), monomethoxy polyethylene glycol - imidazolyl - carbonyl (MePEG-IM), and place of the terminal methoxy group (Eg, HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH ₂ ).

  The PEG portion of the PEG-lipid conjugates described herein can include an average molecular weight ranging from 550 daltons to 10,000 daltons. In certain cases, the PEG moiety is 750-5,000 daltons (eg, 1,000-5,000 daltons, 1,500-3,000 daltons, 750-3,000 daltons, 750 daltons) 〜2,000 daltons). In a preferred embodiment, the PEG moiety has an average molecular weight of 2,000 or 750 daltons.

In certain cases, PEG may be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. PEG can be conjugated directly to the lipid or linked to the lipid via a linker moiety. For example, any linker moiety suitable for attaching PEG to a lipid can be used, including non-ester containing linker moieties and ester containing linker moieties. In a preferred embodiment, the linker moiety is a non-ester containing linker moiety. Suitable non-ester containing linker moieties include amide (-C (O) NH-), amino (-NR-), carbonyl (-C (O)-), carbamate (-NHC (O) O-), urea (-NHC (O) NH-), disulphide (-S-S-), ether (-0-), succinyl _{(- (0) CCH 2 CH} 2 C (0) -), Sukushin'amijiru (-NHC (0) _{CH 2 CH 2 C (0)} NH-), ether, disulphide, and the like linker containing both of these combinations (carbamate linker moiety and an amido linker moiety) include, but are not limited to. In a preferred embodiment, the PEG is attached to the lipid using a carbamate linker.

  In other embodiments, an ester-containing linker moiety is used to attach PEG to a lipid. Suitable ester-containing linker moieties include, for example, carbonate (-OC (O) O-), succinoyl, phosphate (-O- (O) POH-O-), sulfonate, and combinations thereof. Can be

Phosphatidylethanolamines with various acyl chain groups of various chain lengths and degrees of saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamine containing saturated or unsaturated fatty acids with carbon chain lengths in the range of C ₁₀ -C ₂₀ are preferred. Phosphatidylethanolamines having mono- or di-unsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can also be used. Suitable phosphatidylethanolamines include dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE). However, it is not limited to these.

The term “diacylglycerol” or “DAG” includes compounds having ^two aliphatic acyl chains R ¹ and R ² , both of which are independently linked to the glycerol at positions 1 and 2 by an ester linkage. It has from 2 to 30 carbons. Acyl groups can be saturated or have different degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C ₁₂ ), myristoyl (C ₁₄ ), palmitoyl (C ₁₆ ), stearoyl (C ₁₈ ), and icosoyyl (C ₂₀ ). In a preferred embodiment, R ¹ and R ² are the same, ie, R ¹ and R ² are both myristoyl (ie, dimyristoyl), and R ¹ and R ² are both stearoyl (ie, distearoyl).

The term “dialkyloxypropyl” or “DAA” includes compounds with _two alkyl chains R ₁ and R ₂ , both of which independently have 2 to 30 carbons. Alkyl groups can be saturated or have different degrees of unsaturation.

Preferably, PEG-DAA conjugates, PEG-di-decyloxypropylamine _{(C 10)} complex, PEG-dilauryloxypropyl propyl _{(C 12)} complex, PEG-dimyristyloxypropyl _{(C 14)} complex, PEG - dipalmityl propyl _{(C 16)} complex, or PEG- distearyl propyl _{(C 18)} is a composite. In these embodiments, the PEG preferably has an average molecular weight of 750 or 2,000 daltons. In certain embodiments, the terminal hydroxyl group of PEG is replaced with a methyl group.

  In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and Derivatized celluloses such as, but not limited to, hydroxymethylcellulose or hydroxyethylcellulose.

  In some embodiments, the lipid conjugate (eg, PEG-lipid) comprises 0.1 mol% to 2 mol%, 0.5 mol% to 2 mol%, 1 mol% to 2 mol%, 0 mol% to 2 mol% of the total lipid present in the particles. 1.6 mol% to 1.9 mol%, 0.7 mol% to 1.8 mol%, 0.8 mol% to 1.7 mol%, 0.9 mol% to 1.6 mol%, 0.9 mol% to 1.8 mol%, 1 mol % To 1.8 mol%, 1 mol% to 1.7 mol%, 1.2 mol% to 1.8 mol%, 1.2 mol% to 1.7 mol%, 1.3 mol% to 1.6 mol%, or 1.4 mol% １．５1.5 mol% (or an arbitrary ratio thereof or a range thereof). In other embodiments, the lipid conjugate (eg, PEG-lipid) comprises 0 mol% to 20 mol%, 0.5 mol% to 20 mol%, 2 mol% to 20 mol%, 1.5 mol% of the total lipid present in the particle. -18 mol%, 2 mol%-15 mol%, 4 mol%-15 mol%, 2 mol%-12 mol%, 5 mol%-12 mol%, or 2 mol% (or any ratio or range thereof).

  In a further embodiment, the lipid conjugate (eg, PEG-lipid) comprises 4 mol% to 10 mol%, 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol% of the total lipid present in the particle, 6 mol% to 9 mol%, 6 mol% to 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any ratio or range thereof).

  The proportion of lipid conjugate (eg, PEG-lipid) present in the lipid particles of the invention is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, ± 2 mol%. . One skilled in the art will appreciate that the concentration of the lipid complex may vary depending on the lipid complex used and the rate at which the lipid particles become fusogenic.

  By controlling the composition and concentration of the lipid complex, the rate at which the lipid complex exchanges out of the lipid particles, as well as the rate at which the lipid particles become fusogenic, can be controlled. In addition, other variables including, for example, pH, temperature, or ionic strength can be used to change and / or control the rate at which the lipid particles become fusogenic. Other methods that can be used to control the rate at which the lipid particles become fusogenic will become apparent to those of skill in the art upon reading this disclosure. Further, by controlling the composition and concentration of the lipid complex, the particle size of the lipid can be controlled.

  Compositions and formulations for administration

  The nucleic acid-lipid compositions of the present disclosure can be administered by various routes, for example, to provide systemic delivery via intravenous, parenteral, intraperitoneal, or local routes. In some embodiments, the siRNA can be delivered intracellularly, for example, in cells of a target tissue, such as lung or liver, or in inflamed tissue. In some embodiments, the present disclosure provides a method for delivering siRNA in vivo. The nucleic acid-lipid composition can be administered to a subject intravenously, subcutaneously, or intraperitoneally. In some embodiments, the disclosure provides a method for in vivo delivery of interfering RNA to the lungs of a mammalian subject.

  In some embodiments, the disclosure provides a method of treating a disease or disorder in a mammalian subject. A composition of the present disclosure containing a therapeutically effective amount of a core, cationic lipid, amphiphile, phospholipid, cholesterol, and PEG-linked cholesterol can be reduced, reduced, down-regulated, or silenced by the composition. It can be administered to a subject having a disease or disorder associated with gene expression or overexpression.

  The compositions and methods of the present disclosure can be used in a variety of ways, including oral, rectal, vaginal, intranasal, pulmonary, or transdermal or transdermal delivery, or topical delivery to the eye, ear, skin, or other mucosal surface. It can be administered to a subject by a mucosal administration mode. In some aspects of the disclosure, the mucosal tissue layer comprises an epithelial cell layer. Epithelial cells can be lung, trachea, bronchi, alveoli, nose, cheeks, epidermis, or gastrointestinal tract. The compositions of the present disclosure can be administered using conventional actuators, such as mechanical spray devices, as well as pressurized, electrically actuated, or other types of actuators.

  The compositions of the present disclosure may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in a spray form by various methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is achieved by administering the compositions in the form of drops, particles, or sprays, which can be, for example, aerosolized, nebulized, or atomized. The particles of the composition, spray, or aerosol can be in either liquid or solid form. A preferred system for dispensing liquid as a nasal spray is disclosed in U.S. Patent No. 4,511,069. Such formulations can be conveniently prepared by dissolving the composition according to the present disclosure in water to form an aqueous solution and sterilizing the solution. The formulation may be presented in a multi-dose container, for example, in the sealed dispensing system disclosed in US Pat. No. 4,511,069. Another suitable nasal spray delivery system is TRANSDERMAL SYSTEM MEDICATION, Y. W. Chien ed. , Elsevier Publishers, New York, 1985 and U.S. Patent No. 4,778,810. Additional aerosol delivery forms can include, for example, compressed air, jets, ultrasound, and piezoelectric nebulizers, which are dissolved or suspended in a pharmaceutical solvent such as water, ethanol, or mixtures thereof. Deliver a biologically active agent.

  The nasal and pulmonary spray solutions of the present disclosure are typically drug or, if desired, surfactants such as non-ionic surfactants (eg, polysorbate-80) and one or more buffers. And drugs formulated with it. In some embodiments of the present disclosure, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be between pH 6.8 and 7.2. The pharmaceutical solvent used may also be a slightly acidic aqueous buffer of pH 4-6. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases.

  In some embodiments, the present disclosure is a pharmaceutical product that includes a solution containing the composition of the present disclosure and an actuator for lung, mucosal, or intranasal spray or aerosol.

  The dosage form of the composition of the present disclosure can be a liquid in the form of droplets or an emulsion, or in the form of an aerosol.

  The dosage form of the composition of the present disclosure can be a solid that can be reconstituted in a liquid prior to administration. Solids can be administered as a powder. The solid may be in the form of a capsule, tablet, or gel.

  To formulate compositions for pulmonary delivery within the present disclosure, the biologically active agent may be combined with various pharmaceutically acceptable excipients, as well as a base or carrier for dispersion of the active agent (s). Can be combined with Examples of additives include pH adjusters such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (eg, benzyl alcohol), isotonic agents (eg, sodium chloride, mannitol, sorbitol), adsorption inhibitors (eg, Tween 80), solubility enhancers (eg, cyclodextrin and Derivatives thereof), stabilizers (eg, serum albumin), and reducing agents (eg, glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the formulation, when measured relative to the tonicity of 0.9% (w / v) saline, considered unity, is typically: Adjusted to a value that does not cause substantial irreversible tissue damage in the mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of 1/3 to 3, more typically 1/2 to 2, and most often 3/4 to 1.7.

  The biologically active agent can be dispersed in a base or vehicle, which can include a hydrophilic compound capable of dispersing the active agent and any desired additives. The base may be a copolymer of a polycarboxylic acid or a salt thereof, a copolymer of a carboxylic anhydride (eg, maleic anhydride) and another monomer (eg, methyl (meth) acrylate, acrylic acid, etc.), a hydrophilic vinyl polymer, For example, polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose and the like, and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and non-toxic metal salts thereof A wide variety of suitable carriers can be selected including, but not limited to, and the like. Often, biodegradable polymers such as polylactic acid, poly (lactic-co-glycolic acid) copolymer, polyhydroxybutyric acid, poly (hydroxybutyric-co-glycolic acid) copolymer, and mixtures thereof, are selected as the base or carrier. You. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters and sucrose fatty acid esters can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and can impart enhanced structural integrity to the carrier by partial crystallization, ionic bonding, crosslinking, and the like. The carrier can be provided in various forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of a selected carrier in this context can result in enhanced absorption of the biologically active agent.

  Formulations for mucosal, nasal, or pulmonary delivery may contain a hydrophilic low molecular weight compound as a base or excipient. Such hydrophilic low molecular weight compounds provide a passage medium through which a water-soluble active agent, such as a physiologically active peptide or protein, can diffuse through the base to the body surface where the active agent is absorbed. The hydrophilic low molecular weight compound, if desired, absorbs water from the mucosa or the atmosphere of administration and dissolves the water-soluble active peptide. The molecular weight of the hydrophilic low molecular weight compound is generally 10,000 or less, preferably 3,000 or less. Examples of hydrophilic low molecular weight compounds include sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, polyethylene glycol And polyol compounds such as oligosaccharides, disaccharides and monosaccharides, including mixtures thereof. Further examples of hydrophilic low molecular weight compounds include N-methylpyrrolidone, alcohols (eg, oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

  Compositions of the present disclosure may alternatively include pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate. , And mixtures thereof, and may contain as carrier a pharmaceutically acceptable substance required to approximate physiological conditions. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers are used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. can do.

  In certain embodiments of the present disclosure, the biologically active agent can be administered in a time release formulation, such as a composition that includes a sustained release polymer. The active agent can be prepared with carriers that will protect against rapid release, such as a controlled release vehicle, such as a polymer, microencapsulated delivery system, or bioadhesive gel. Prolonged delivery of the active agents in various compositions of the present disclosure can be provided by including in the composition an agent that delays absorption, for example, aluminum monooterate hydrogel and gelatin.

  Although the present disclosure has been described with respect to certain specific embodiments, and numerous details are set forth for purposes of illustration, the present disclosure includes additional embodiments and may be used without departing from the present disclosure. It will be apparent to those skilled in the art that some of the details described in the written description may be significantly varied. The present disclosure includes such additional embodiments, modifications, and equivalents. In particular, this disclosure includes any combination of features, terms, or elements of various exemplary components and embodiments.

最初の数の対は、Ｌ_１及びＬ_２について、カルボニルを含むエステル中の炭素数を示し、括弧内の数の対は、分枝状アルキルＲ_１、又はＲ_２も分枝状である場合、Ｒ_１／Ｒ_２（星印は二重結合を示す）の各分枝中の炭素数を示し、Ｒ_３の炭素数が示され、最後の行はＲ_４及びＲ_５の置換を示す。ＡＴＸの番号は、本明細書で参照するために示される。計算されたＬｏｇＤ値（ｃ−ＬｏｇＤ）及び計算されたｐＫａ（ｃ−ｐＫａ）値、並びに括弧内の測定されたｐＫａ（製剤環境下で測定された）が示される。ｃ−ＬｏｇＤ及びｃ−ｐＫａ値は、ＡＣＤ Ｌａｂｓ Ｓｔｒｕｃｔｕｒｅ Ｄｅｓｉｇｎｅｒ ｖ１２．０．により生成される。生物活性は、別段の指定がない限り、０．０３ｍｇ／ｋｇの用量でのインビボ第ＶＩＩ因子ノックダウン率である。

Example 1 Exemplary Lipids Exemplary compounds of Formula I are provided in Table 1. The structure of the compound is shown in the first column. The designation of the compounds is given according to formula I.

The first number pair indicates, for L ₁ and L ₂ , the number of carbon atoms in the ester containing the carbonyl; the number pair in parentheses indicates that the branched alkyl R ₁ or R ₂ is also branched. , R ₁ / R ₂ (the asterisk indicates a double bond) indicates the number of carbons in each branch, R ₃ indicates the number of carbons, and the last row indicates the substitution of R ₄ and R ₅ . ATX numbers are provided for reference herein. The calculated LogD value (c-LogD) and the calculated pKa (c-pKa) value, and the measured pKa in parentheses (measured in the formulation environment) are shown. c-LogD and c-pKa values are based on ACD Labs Structure Designer v12.0. Generated by Biological activity is the in vivo factor VII knockdown rate at a dose of 0.03 mg / kg unless otherwise specified.

  Example 2: Synthesis of ATX-0043

  FIG. 1 shows the synthesis route for ATX-0043, which is described further below.

ATX-0043: Step 1

  A 500 mL single neck round bottom flask was charged with 25 g of hexanoic acid (SM1; 1 eq.) Dissolved in dichloromethane (DCM; 200 mL) and then 27.6 mL of oxalyl chloride at 0 ° C. with stirring under a nitrogen atmosphere. (1.5 eq) was added slowly, followed by 0.5 mL of dimethylformamide (DMF; catalyst). The resulting reaction mixture was stirred at room temperature for 2 hours.

In a separate 1-liter two-necked round-bottom flask, 31.4 g of N, O-dimethylhydroxylamine hydrochloride (1.5 eq) in DCM (200 mL) was stirred at 0 ° C. using an addition funnel. 89.8 mL of triethylamine (Et ₃ N, 3 eq) was added. After concentration under reduced pressure to the resulting solution, the acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (100 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by thin layer chromatography (TLC) (20% ethyl acetate (EtOAc) / hexane; Rf: 0.5). The reaction mass was diluted with water (300 mL). The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography using (60-120 silica gel; 10% EtOAc / hexane). 20.0 g produced; 58% yield.

ATX-0043: Step 2

  To a solution of 33 g of pentylmagnesium bromide (1.5 eq) in tetrahydrofuran (THF; 100 mL), placed in a 500 mL two-necked round bottom flask and stirred at 0 ° C. under a nitrogen atmosphere, 20 g of N-methoxy-N -Methylhexanamide (1 eq) solution (dissolved in 200 mL of THF) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with a saturated NH ₄ Cl solution (150 mL) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / hexane). Production amount 15.0 g; yield 66%.

ATX-0043: Step 3

  To a solution of 15 g undecane-6-one (1 equiv) dissolved in 25 mL methanol (MeOH) in 150 mL THF at 0 ° C. was added 4.9 g sodium borohydride (1.5 equiv) to give The resulting solution was stirred at room temperature for 2 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (100 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (150 mL) and water (150 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. Production amount 14.0 g; yield 93%.

ATX-0043: Step 4

  To a solution of 15 g of 4-aminobutanoic acid (1 equiv.) Dissolved in 150 mL of THF at 0 ° C. was added 145 mL of aqueous 1N NaOH solution (1 equiv.), Followed by 43.4 mL of Boc anhydride (1.3 equiv.). Were added sequentially using an addition funnel over a 15 minute period. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% MeOH in chloroform (CHCl ₃ ); Rf: 0.5). The reaction mass was quenched with 5% HCl (150 mL) and then EtOAc (100 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. Production amount 20.0 g; yield 68%.

ATX-0043: Step 5

To a solution of 12 g of 4-((tert-butoxycarbonyl) amino) butanoic acid (1 equivalent) dissolved in DCM (200 mL) and cooled below 0 ° C., was added 14.7 g of 1-ethyl-3- (3 -Dimethylaminopropyl) carbodiimide (EDC). HCl (1.3 eq), 10.6 mL of _Et 3 N (1.3 eq), and 0.72g of 4-dimethylaminopyridine; the (DMAP 0.1 eq) under a nitrogen atmosphere, at 10 minute intervals It was added sequentially. To the resulting solution was added alcohol at the same temperature by dissolving in DCM (50 mL) using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with water (100 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x50mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (100 mL) and then extracted with EtOAc (2 × 50 mL). The organic layer was concentrated under reduced pressure and the crude was taken to the next step. 8.5 g produced; 48% yield.

ATX-0043: Step 6

  To a solution of 8.5 g undecane-6-yl 4-((tert-butoxycarbonyl) amino) butanoate (1 eq) dissolved in 70 mL DCM at 0 ° C. was added trifluoroacetic acid (TFA; 10 eq). The mixture was stirred at room temperature for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (70% EtOAc / hexane; Rf: 0.2). The reaction mass was concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then extracted with EtOAc (2 × 100 mL). The organic layer was separated and concentrated under reduced pressure.

The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol. Production 5.0 g; yield 33% (based on alcohol).

ATX-0043: Step 7

A solution of 14 g of 4-bromobutyric acid (1 equivalent) dissolved in DCM (100 mL) and cooled below 0 ° C. was added with 21 g of EDC. HCl (1.3 eq), 15.2 mL of _Et 3 N (1.3 eq), and 1g of DMAP (0.1 eq) under a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution, using an addition funnel, add 8.3 g of (Z) -non-2-en-1-ol (0.7 eq) by dissolving in 50 mL of DCM and adding a nitrogen atmosphere. Stirred at room temperature for 16 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with water (50 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x50mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (100 mL) and then extracted with EtOAc (2 × 50 mL). The organic layer was separated and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / hexane. Production amount 11.0 g; yield 64%.

ATX-0043: Step 8

  In a 250 mL round bottom flask, 2 g of undecane-6-yl 4-aminobutanoate (1 equivalent) and 2.2 g of (Z) -non-2-en-1-yl 4-bromobutanoate in DMF (1 equiv), 1.2 g of potassium carbonate (1.2 equiv) were added and the resulting mixture was refluxed at 90 ° C. for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . Ice water was added to the reaction mass, then extracted with EtOAc, dried over sodium sulfate and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / hexane. The starting amine and bromo compound were recovered. 1.45 g produced; 40% yield.

ATX-0043: Step 9

  A solution of 1.45 g of (Z) -non-2-en-1-yl 4-((4-oxo-4- (undecane-6-yloxy) butyl) amino) butanoate (1 equivalent) dissolved in dry DCM To this was added 1.29 mL of triethylamine (3 eq) and 360 mg of triphosgene (0.4 eq) at 0 ° C. under nitrogen atmosphere at 5 min intervals. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, in a two-necked 250 mL round bottom flask stirred at 0 ° C. in 360 mg of sodium hydride (5.5 equiv.) Dissolved in dry THF (20 mL) was added 2.1 g of 2-hexane in THF (30 mL). (Dimethylamino) ethane-1-thiol hydrochloride (5.5 eq) was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To the resulting solution was slowly added the above carbonyl chloride dissolved in THF (50 mL) using an addition funnel over about 15 minutes, added to the resulting solution and stirred at room temperature for 1 hour.

The reaction mass was quenched with a saturated NH ₄ Cl solution (20 mL) and then EtOAc (20 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 20 mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography. The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA carbonization).

Purification was performed using silica gel (100-200 mesh; 18% EtOAc / hexane) chromatography. 500 mg; yield 26%; confirmed by ¹ H NMR; HPLC; and mass spectrometry (mass).

ATX-0043 / RL-43A: ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 5.63 (m, 1), 5.54 (m, 1), 4.87 (m, 1), 4.63 (d, J = 7.0, 2), 3.37 (brs, 4), 3.03 (t, J = 7.0, 2), 2.27 (s, 6), 2. 22 to 2.32 (4), 2.09 (m, 2), 1.80 to 1.90 (4), 1.45 to 1.55 (4), 1.20 to 1.40 (22) , 0.83-0.92 (9).
Example 3: Synthesis of ATX-0057

  FIG. 2 shows the synthesis route for ATX-0057, which is described further below.

ATX-0057: Step 1: N-methoxy-N-methyloctamide

  Octanoic acid (1 eq.) Dissolved in DCM (300 mL) was charged into a 2 liter two-necked round bottom flask, and then 1.5 eq. Oxalyl chloride was added slowly at 0 ° C. with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2-liter two-necked round-bottom flask, to 2 equivalents of N, O-dimethylhydroxylamine hydrochloride in DCM (200 mL) was added 3 equivalents of trimethylamine stirred at 0 ° C. using an addition funnel. . After concentration under reduced pressure to the resulting solution, the above acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (150 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (250 mL). The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / hexane). 85 g; yield 65%.

ATX-0057: Step 2: hexadecane-8-one

  A solution of octylmagnesium bromide in THF (100 mL), stirred in a 1 liter two-necked round bottom flask at 0 ° C. under a nitrogen atmosphere, was added to a N-methoxy-N-methyloctaneamide solution (200 mL of THF). Dissolution) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with a saturated NH ₄ Cl solution (250 mL) and then EtOAc (350 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / hexane). 65 g yield; 63% yield.

ATX-0057: Step 3: hexadecane-8-ol

  To a solution of hexadecane-8-one (1 equivalent) dissolved in MeOH / THF was added 1 equivalent of sodium borohydride at 0 ° C. and the resulting solution was stirred at room temperature for 1.5 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (150 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. Production amount 60 g; yield 91%.

ATX-0057: Step 4: 4-((tert-butoxycarbonyl) amino) butanoic acid

  To a solution of 4-aminobutanoic acid in THF at 0 ° C. was added an aqueous 1N NaOH solution followed by Boc anhydride over 15 minutes using an addition funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was quenched with 5% HCl (250 mL) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x150mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. 80 g; yield 81%.

ATX-0057: Step 5: hexadecane-8-yl 4-((tert-butoxycarbonyl) amino) butanoate

EDC. Was added to a solution of 4-((tert-butoxycarbonyl) amino) butanoic acid dissolved in DCM (200 mL) and cooled below 0 ° C. HCl, _Et 3 N, and 4-dimethylaminopyridine (DMAP) under a nitrogen atmosphere, were successively added at 10 minute intervals. To the resulting solution was added 1 equivalent of hexadecane-8-ol alcohol at the same temperature by dissolving in DCM (150 mL) using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with water (150 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then EtOAc (200 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. 80 g (crude; necessary compounds and alcohols) produced.

ATX-0057: Step 6: hexadecane-8-yl 4-aminobutanoate

To a solution of hexadecane-8-yl-4-((tert-butoxycarbonyl) amino) butanoate dissolved in DCM was added TFA at 0 ° C, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} . The reaction mass was concentrated under reduced pressure. The resulting crude was washed with a saturated NaHCO ₃ solution (300 mL) and then extracted with EtOAc (2 × 200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol. Production amount 40 g; 59% yield in two steps; confirmed by mass.

ATX-0057: Step 7: (Z) -Non-2-en-1-yl 4-bromobutanoate

EDC. Was added to a solution of 4-bromobutyric acid, dissolved in DCM (400 mL) and cooled below 0 ° C. HCl, Et ₃ N, and DMAP were added sequentially at 10 minute intervals under a nitrogen atmosphere. To the resulting solution was added (Z) -non-2-en-1-ol by dissolving in 100 mL DCM using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with water (300 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x150mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (200 mL) and then extracted with EtOAc (150 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / hexane. The alcohol was recovered. Production amount 27 g; yield 51%.

ATX-0057: Step 8: hexadecane-8-yl (Z) -4-((4- (non-2-en-1-yloxy) -4-oxobutyl) amino) butanoate

Potassium carbonate was added to a solution of hexadecan-8-yl 4-aminobutanoate, (Z) -non-2-en-1-yl 4-bromobutanoate in acetonitrile (ACN) and the resulting mixture was obtained. Was refluxed at 90 ° C. for 4 hours under a nitrogen atmosphere. The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was filtered, washed with ACN (20 mL) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / hexane. The starting amine and bromo compounds were recovered. 20 g; yield 40%; confirmed by mass.

ATX-0057: Step 9

  To a solution of hexadecane-8-yl (Z) -4-((4- (non-2-en-1-yloxy) -4-oxobutyl) amino) butanoate dissolved in dry DCM was added trimethylamine and triphosgene in a nitrogen atmosphere. Add below at 0 ° C. at 5 minute intervals. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, 2- (dimethylamino) propane-1-thiol hydrochloride was added to sodium hydride dissolved in dry THF (50 mL) in a two-neck 100 mL round bottom flask stirred at 0 ° C. Stirring was continued for 5 minutes below. To the resulting solution, the carbamoyl chloride, dissolved in THF (80 mL), was slowly added via syringe over about 10 minutes. The resulting solution was stirred at room temperature for 6 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL) and then EtOAc (150 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x40mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

Initial purification was performed using silica gel (60-120 mesh), 22 g of the crude compound was adsorbed on 60 g of silica gel and poured onto 500 g of silica gel placed in a column. Compound was eluted with 35% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. 7.5 g of the crude compound was adsorbed on 18 g of neutral alumina and the resulting was poured onto 130 g of neutral alumina placed in a column. Compound eluted with 10% EtOAc / hexane. Yield 29%; confirmed by ¹ H NMR, HPLC, and mass.

ATX-0057 / RL-43C: ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 5.63 (m, 1), 5.51 (m, 1), 4.68 (m, 1), 4.83 (d, J = 7.0, 2), 3.19 (brs, 4), 3.22 (m, 2), 2.52 (m, 2), 2.23 to 2.37 ( 4), 2.18 (s, 6), 2.08 (m, 2), 1.84 to 1.93 (4), 1.46 to 1.54 (4), 1.20 to 1.40 (30), 0.83-0.91 (9).
Example 4: Synthesis of ATX-0058

  FIG. 3 shows the synthesis route for ATX-0058, which is described further below.

ATX-0058: Step 1

During two-necked round bottom flask 500mL under N ₂ atmosphere, placed in 30g dissolved in DCM and 200 mL 8- bromooctanoate (1 eq), followed by stirring under a nitrogen atmosphere, 26.7 mL at 0 ℃ Was slowly added to oxalyl chloride (1.5 eq). The resulting reaction mixture was stirred at room temperature for 2 hours.

  In a separate 1 liter two-necked round bottom flask, add 40.5 g of N, O-dimethylhydroxylamine hydrochloride (2 eq) in 300 mL of DCM and add 87 mL of trimethylamine (3 eq) stirred at 0 ° C. did. After concentration under reduced pressure to the resulting solution, the acid chloride was added dropwise by dissolving in 500 mL of DCM over 15 minutes using an addition funnel. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (300 mL). The organic layer was separated and the aqueous layer was washed with DCM (2x100mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / hexane). Generated amount 28g.

ATX-0058: Step 2

  To a solution of 28 g of hexylmagnesium bromide (1 equivalent) in THF (100 mL), stirred at 0 ° C. under a nitrogen atmosphere, was added 36.8 g of N-methoxy-N-methyloctaneamide (1.3 mL) in 200 mL of THF. Eq.) And the resulting reaction mixture was stirred at room temperature for 5 hours.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.7). The reaction mass was quenched with a saturated NH ₄ Cl solution (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% ethyl acetate / hexane). 24 g; yield 77%.

ATX-0058: Step 3

  To a solution of 24 g of tetradecane-7-one (1 equivalent) dissolved in MeOH / THF at 0 ° C. was added 4.27 g of sodium borohydride (1 equivalent) and the resulting solution was stirred at room temperature for 1 hour. did.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (50 mL). The methanol was reduced under reduced pressure. The resulting crude was partitioned between EtOAc (200 mL) and water. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 80 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. 21.5 g produced; 89% yield.

ATX-0058: Step 4

  To a solution of 20 g of 4-aminobutanoic acid in 140 mL of THF at 0 ° C. was added 196 mL of an aqueous 1N NaOH solution, followed by 36.8 g of Boc anhydride using a funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.5). The reaction mass was quenched with 5% HCl (100 mL) and then EtOAc (200 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. Production amount 30 g; yield 76%.

ATX-0058: Step 5

To a solution of 10 g of 4-((tert-butoxycarbonyl) amino) butanoic acid (1 equivalent) dissolved in DCM (150 mL) and cooled below 0 ° C. was added 12.2 g of EDC. HCl (1.3 equiv), _Et 3 N (3 eq) in 20.4 mL, and 488mg of DMAP (0.1 eq) were sequentially added at 10 minute intervals. To the resulting solution was added an alcohol by dissolving in DCM using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mass was quenched with water (100 mL) and the organic layer was separated. The aqueous layer was washed with DCM (2x50mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution and EtOAc (100 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. Production amount 12.7 g (crude).

ATX-0058: Step 6

  To a solution of 12.5 g of tetradecane-7-yl 4-((tert-butoxycarbonyl) amino) butanoate (1 eq) dissolved in 100 mL of DCM at 0 ° C. was added 23.9 mL of TFA (10 eq). The mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (100 mL) and EtOAc (100 mL) was added. The organic layer was separated and concentrated under reduced pressure.

The crude compounds; subjected to column chromatography using (60-120 mesh silica gel 4% MeOH / CHCl _3), to recover the alcohol. 7 g produced in two steps; 47% yield; confirmed by mass.

ATX-0058: Step 7

A solution of 20 g of 4-bromobutyric acid (1 equivalent) dissolved in DCM (150 mL) and cooled to 0 ° C. was added with 1.5 equivalents of EDC. HCl, 3 eq. Et ₃ N, and 0.1 eq. DMAP were added sequentially at 10 minute intervals. To this resulting solution is added, using a funnel, 0.7 equivalents of (Z) -non-2-en-1-ol by dissolving in 100 mL of DCM and adding 24 hours at room temperature under a nitrogen atmosphere. Stirred for hours.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.7). The reaction mass was quenched with water (100 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution and EtOAc (150 mL) was added. The organic layer was separated and concentrated under reduced pressure.

The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 5% EtOAc / hexane). 17 g; yield 69%; confirmed by ¹ H NMR.

ATX-0058: Step 8

  6 g tetradecane-7-yl 4-aminobutanoate (1 equivalent) in ACN (125 mL), 5.8 g (Z) -non-2-en-1-yl 4-bromobutanoate (1 equivalent) 2.7 g of potassium carbonate (1.2 equivalents) was added to the solution of the above, and the resulting mixture was refluxed at 90 ° C. for 3 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.5). The reaction mass was filtered and the filtrate was concentrated under reduced pressure.

  The crude compound was subjected to column chromatography using (100-200 mesh silica gel; 15% EtOAc / hexane). 4.5 g; yield 44%; confirmed by mass.

ATX-0058: Step 9

  4.4 g of (Z) -non-2-en-1-yl 4-((4-oxo-4- (tetradecane-7-yloxy) butyl) amino) butanoate (1 equivalent) dissolved in 30 mL of dry DCM To a solution of was added 0.83 mL of trimethylamine (3 eq) and 418 mg of triphosgene (0.5 eq) at 0 ° C. under a nitrogen atmosphere at 5 min intervals. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  To 192 mg of sodium hydride (10 eq) dissolved in dry THF (25 mL) in a two-neck 100 mL round bottom flask was added 564 mg of 2- (dimethylamino) propane-1-thiol hydrochloride (5 eq) at 0 ° C. The addition was continued with stirring for 5 minutes under a nitrogen atmosphere. To the resulting solution, the carbamoyl chloride, dissolved in THF (35 mL), was added slowly via syringe over about 10 minutes. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 4 hours.

The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with saturated NH ₄ Cl (30 mL) and then EtOAc (100 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 50 mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

The first purification was performed using silica gel (60-120 mesh). 5.0 g of the crude compound was adsorbed on 9 g of silica gel and poured onto 90 g of silica gel placed in a column. Compound was eluted with 35% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. 1.5 g of the crude compound was adsorbed on 4 g of neutral alumina, and the resultant was poured on 40 g of neutral alumina placed in a column. Compound eluted with 10% EtOAc / hexane. 1.2 g; yield 21%; confirmed by ¹ H NMR; HPLC; mass.

ATX-0058 / RL-43B: ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 5.65 (m, 1), 5.52 (m, 1), 4.86 (m, 1), 4.63 (d, J = 7.0, 2), 3.37 (brs, 4), 3.02 (t, J = 6.0, 2), 2.53 (t, J = 6.0) , 2), 2.27 to 2.36 (4), 2.27 (s, 6), 2.09 (m, 2), 1.83 to 1.96 (4), 1.46 to 1. 54 (4), 1.20 to 1.40 (26), 0.84 to 0.91 (9).
Example 5: Synthesis of ATX-0081

  FIG. 4 shows the synthesis route for ATX-0081, which is described further below.

ATX-0081: Step 1

  Octanolic acid dissolved in DCM (200 mL) was charged into a 2 liter two-necked round bottom flask, and then 1.5 equivalents of oxalyl chloride was slowly added at 0 ° C. with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2-liter two-necked round-bottom flask, to 2 equivalents of N, O-dimethylhydroxylamine hydrochloride in DCM (200 mL) was added 3 equivalents of trimethylamine stirred at 0 ° C. using an addition funnel. . After concentration under reduced pressure to the resulting solution, the above acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (150 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (250 mL). The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / hexane). 33 g; 84% yield.

ATX-0081: Step 2

  To a solution of 22 g of heptylmagnesium bromide (1.5 eq) in THF (100 mL), stirred in a 1 liter two-necked round bottom flask at 0 ° C. under nitrogen atmosphere, was added N-methoxy-N-methyloctane. An amide (1 equiv) solution (dissolved in 200 mL of THF) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with a saturated NH ₄ Cl solution (250 mL) and then EtOAc (350 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / hexane). Production amount 22 g; yield 65%.

ATX-0081: Step 3

  To a solution of 22 g of pentadec-8-one (1 equivalent) dissolved in MeOH / THF was added 1.5 equivalents of sodium borohydride at 0 ° C. and the resulting solution was stirred at room temperature for 1 hour.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (150 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. Production amount 20 g; yield 90%.

ATX-0081: Step 4

  To a solution of 50 g of 4-aminobutanoic acid in 350 mL of THF at 0 <0> C was added 490 mL of aqueous 1 N NaOH solution, followed by 140 mL of Boc anhydride sequentially over 15 minutes using an addition funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was quenched with 5% HCl (250 mL) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x150mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. 80 g; yield 81%.

ATX-0081: Step 5

To a solution of 10 g of 4-((tert-butoxycarbonyl) amino) butanoic acid dissolved in DCM (250 mL) and cooled below 0 ° C., 1.3 equivalents of EDC. HCl, _Et 3 N, and 4-dimethylaminopyridine (DMAP), in a nitrogen atmosphere, were successively added at 10 minute intervals. To the resulting solution was added 1 equivalent of pentadec-7-ol alcohol at the same temperature by dissolving in DCM (150 mL) using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with water (150 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then EtOAc (200 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. 8.5 g (crude; required compounds and alcohols) produced.

ATX-0081: Step 6

  To a solution of 8.5 g of pentadecane-8-yl 4-((tert-butoxycarbonyl) amino) butanoate in 65 mL of DCM was added 10 equivalents of TFA at 0 ° C and 3 hours at room temperature under nitrogen atmosphere. Stirred.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} . The reaction mass was concentrated under reduced pressure. The resulting crude was washed with a saturated NaHCO ₃ solution (300 mL) and then extracted with EtOAc (2 × 200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol. 4 g produced in two steps; yield 25%; confirmed by mass.

ATX-0081: Step 7

A solution of 4-bromobutyric acid, dissolved in DCM (300 mL) and cooled below 0 ° C., was charged with EDC. HCl, Et ₃ N, and DMAP were added sequentially at 10 minute intervals under a nitrogen atmosphere. To this resulting solution, using an addition funnel, add 20 g of (Z) -non-2-en-1-ol by dissolving in 100 mL of DCM and stirring at room temperature under a nitrogen atmosphere for 24 hours. did.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with water (300 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x150mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (200 mL) and then extracted with EtOAc (150 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / hexane. The alcohol was recovered. 19 g; 55% yield.

ATX-0081: Step 8

  To a solution of 4.5 g pentadecane-8-yl 4-aminobutanoate, 1 equivalent of (Z) -non-2-en-1-yl 4-bromobutanoate in 70 mL acetonitrile (ACN), 0.4 equivalents of potassium carbonate were added and the resulting mixture was refluxed at 90 ° C. for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was filtered, washed with ACN (20 mL) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / hexane. The starting amine and bromo compounds were recovered. Produced amount 2.1 g; yield 27%; confirmed by mass.

ATX-0081: Step 9

  To a solution of 2.1 g pentadecan-8-yl (Z) -4-((4- (non-2-en-1-yloxy) -4-oxobutyl) amino) butanoate in 150 mL dry DCM was added Equivalent amounts of triethylamine and triphosgene were added at 0 ° C. at 5 minute intervals under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, 3.5 equivalents of 2- (dimethylamino) propane-1-thiol was added to 7 equivalents of sodium hydride dissolved in dry THF (80 mL) in a two-neck 100 mL round bottom flask stirred at 0 ° C. The hydrochloride was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To the resulting solution, the carbamoyl chloride, dissolved in THF (80 mL), was slowly added via syringe over about 10 minutes. The resulting solution was stirred overnight at 0 ° C. to room temperature under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL) and then EtOAc (150 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x40mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

Initial purification was performed using silica gel (60-120 mesh), the crude compound was adsorbed on 60 g silica gel and poured onto 500 g silica gel in a column. Compound was eluted with 35% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. The crude compound was adsorbed on 18 g of neutral alumina and the resultant was poured on 130 g of neutral alumina placed in a column. Compound eluted with 10% EtOAc / hexane. 1.5 g; yield 45%; confirmed by ¹ H NMR, HPLC, and mass.

ATX-0081 / RL-48B: ¹ H-NMR (PPM, 500 MHz, CDCl ₃ ): δ = 5.64 (m, 1), 5.52 (m, 1), 4.86 (m, 1), 4.63 (d, J = 7.0, 2), 3.31 to 3.44 (4), 3.02 (t, J = 7.0, 2), 2.52 (t, J = 7) 2.0, 2.), 2.26 to 2.36 (4), 2.27 (s, 6), 2.10 (m, 2), 1.84 to 1.95 (4), 1.46 to 1.54 (4), 1.20 to 1.40 (26), 0.85 to 0.94 (9).
Example 6: Synthesis of ATX-0082

  FIG. 5 shows the synthesis route for ATX-0082, which is described further below.

ATX-0082: Step 1

  A 2-liter two-necked round bottom flask was charged with 30 g of octanoic acid dissolved in DCM (200 mL), and then 1.5 eq. Of oxalyl chloride was slowly added at 0 ° C. with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2-liter two-necked round-bottom flask, to 2 equivalents of N, O-dimethylhydroxylamine hydrochloride in DCM (200 mL) was added 3 equivalents of trimethylamine stirred at 0 ° C. using an addition funnel. . After concentration under reduced pressure to the resulting solution, the above acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (150 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (250 mL). The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / hexane). 33 g; 84% yield.

ATX-0082: Step 2

  To a solution of heptylmagnesium bromide (1.5 eq) in THF (100 mL), stirred in a 1 liter two-necked round bottom flask at 0 ° C. under a nitrogen atmosphere, 28 g of N-methoxy-N-methyl Octaneamide (1 equivalent) solution (dissolved in 200 mL of THF) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with a saturated NH ₄ Cl solution (250 mL) and then EtOAc (350 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / hexane). Production amount 22 g; yield 65%.

ATX-0082: Step 3

  To a solution of 22 g of pentadec-8-one (1 equivalent) dissolved in MeOH / THF was added 1.5 equivalents of sodium borohydride at 0 ° C. and the resulting solution was stirred at room temperature for 1 hour.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (150 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. Production amount 20 g; yield 90%.

ATX-0082: Step 4

  To a solution of 15 g of 4-aminobutanoic acid in 120 mL of THF at 0 ° C. was added 185 mL of aqueous 1N NaOH solution, followed by 50 mL of Boc anhydride sequentially over 15 minutes using an addition funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was quenched with 5% HCl (250 mL) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x150mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. 27 g; yield 85%.

ATX-0082: Step 5

To a solution of 10 g of 4-((tert-butoxycarbonyl) amino) butanoic acid dissolved in DCM (250 mL) and cooled below 0 ° C., 1.3 equivalents of EDC. HCl, _Et 3 _N, and 4-dimethylaminopyridine (DMAP), in a nitrogen atmosphere, were successively added at 10 minute intervals. To the resulting solution was added 1 equivalent of pentadec-7-ol alcohol at the same temperature by dissolving in DCM (150 mL) using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with water (150 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then EtOAc (200 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. 8 g of product (crude; required compounds and alcohols).

ATX-0082: Step 6

  To a solution of 8.0 g of pentadecane-8-yl 4-((tert-butoxycarbonyl) amino) butanoate dissolved in 60 mL of DCM at 0 ° C. was added 10 equivalents of TFA and 3 hours at room temperature under nitrogen atmosphere. Stirred.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} . The reaction mass was concentrated under reduced pressure. The resulting crude was washed with a saturated NaHCO ₃ solution (300 mL) and then extracted with EtOAc (2 × 200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol. 4 g; yield 25% in two steps; confirmed by mass.

ATX-0082: Step 7

To a solution of 4-bromobutyric acid dissolved in DCM (400 mL) and cooled below 0 ° C. was added 1.5 equivalents of EDC. HCl, 3 equivalents of _Et 3 N, and DMAP, in a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution, using an addition funnel, add 20 g of (Z) -non-2-en-1-ol by dissolving in 100 mL of DCM and stirring at room temperature under a nitrogen atmosphere for 24 hours. did.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with water (300 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x150mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (200 mL) and then extracted with EtOAc (150 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / hexane. The alcohol was recovered. 18 g; 55% yield.

ATX-0082: Step 8

  In a solution of 4.0 g of pentadecane-8-yl 4-aminobutanoate, 1 equivalent of (Z) -non-2-en-1-yl 4-bromobutanoate in 90 mL of ACN, 1.4 equivalents. Of potassium carbonate was added and the resulting mixture was refluxed at 90 ° C. for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was filtered, washed with ACN (20 mL) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / hexane. The starting material (amine and bromo compound) was recovered. 2.2 g; yield 30%; confirmed by mass.

ATX-0082: Step 9

  To a solution of 2.2 g of pentadecan-8-yl (Z) -4-((4- (non-2-en-1-yloxy) -4-oxobutyl) amino) butanoate in 25 mL of dry DCM was added Equivalent amounts of triethylamine and triphosgene were added at 5 ° C at 0 ° C under nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, 3.5 equivalents of 2- (dimethylamino) propane-1-thiol was added to 7 equivalents of sodium hydride dissolved in dry THF (100 mL) in a two-necked 100 mL round bottom flask stirred at 0 ° C. The hydrochloride was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To the resulting solution, the carbamoyl chloride, dissolved in THF (100 mL), was slowly added via syringe over about 10 minutes. The resulting solution was stirred overnight at 0 ° C. to room temperature under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL) and then EtOAc (150 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x40mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

Initial purification was performed using silica gel (60-120 mesh), the crude compound was adsorbed on 60 g silica gel and poured onto 500 g silica gel in a column. Compound was eluted with 35% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. The crude compound was adsorbed on 18 g of neutral alumina and the resultant was poured on 130 g of neutral alumina placed in a column. Compound eluted with 10% EtOAc / hexane. 1.2 g; yield 43%; confirmed by ¹ H NMR, HPLC, and mass.

ATX-0082 / RL-47A: ¹ H-NMR (PPM, 500 MHz, CDCl ₃ ): δ = 5.64 (m, 1), 5.52 (m, 1), 4.87 (m, 1), 4.62 (d, J = 7.0, 2), 3.61 (t, J = 7.0, 2), 3.28 to 3.37 (2), 3.02 (t, J = 7) .0, 2), 2.61 (m, 2), 2.52 (t, J = 7.0, 2), 2.31 (m, 2), 2.27 (s, 6), 2. 10 (m, 2), 1.62 to 1.70 (6), 1.21 to 1.40 (32), 0.85 to 0.91 (9).
Example 7: Synthesis of ATX-0086

  FIG. 6 shows the synthesis route for ATX-0086, which is described further below.

ATX-0086: Step 1

  A 2-liter two-necked round bottom flask was charged with 30 g of octanoic acid dissolved in DCM (200 mL), and then 1.5 eq. Of oxalyl chloride was slowly added at 0 ° C. with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2-liter two-necked round-bottom flask, to 2 equivalents of N, O-dimethylhydroxylamine hydrochloride in DCM (200 mL) was added 3 equivalents of trimethylamine stirred at 0 ° C. using an addition funnel. . After concentration under reduced pressure to the resulting solution, the above acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (150 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (250 mL). The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / hexane). 38 g; yield 84%; confirmed by mass.

ATX-0086: Step 2

  To a solution of hexylmagnesium bromide (1.5 eq) in THF (100 mL), stirred in a 1 liter two-necked round bottom flask at 0 ° C. under a nitrogen atmosphere, 38 g of N-methoxy-N-methyl Octaneamide (1 equivalent) solution (dissolved in 200 mL of THF) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with a saturated NH ₄ Cl solution (250 mL) and then EtOAc (350 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / hexane). 44 g; yield 65%; confirmed by mass.

ATX-0086: Step 3

  To a solution of 44 g of tridecane-7-one (1 eq) dissolved in MeOH / THF was added 1.5 eq of sodium borohydride at 0 ° C. and the resulting solution was stirred at room temperature for 1 hour.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (150 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. 40 g; 90% yield; confirmed by mass.

ATX-0086: Step 4

  To a solution of 50 g of 4-aminobutanoic acid in 350 mL of THF at 0 <0> C was added 490 mL of aqueous 1 N NaOH solution, followed by 140 mL of Boc anhydride sequentially over 15 minutes using an addition funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was quenched with 5% HCl (250 mL) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x150mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. 80 g; yield 81%; confirmed by mass.

ATX-0086: Step 5

To a solution of 10 g of 4-((tert-butoxycarbonyl) amino) butanoic acid dissolved in DCM (250 mL) and cooled below 0 ° C., 1.3 equivalents of EDC. HCl, _Et 3 _N, and 4-dimethylaminopyridine (DMAP), in a nitrogen atmosphere, were successively added at 10 minute intervals. To the resulting solution was added 1 equivalent of pentadec-7-ol alcohol at the same temperature by dissolving in DCM (150 mL) using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with water (150 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then EtOAc (200 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. 8 g of product (crude; required compounds and alcohols).

ATX-0086: Step 6

  To a solution of 8.0 g of pentadecane-8-yl 4-((tert-butoxycarbonyl) amino) butanoate dissolved in 60 mL of DCM at 0 ° C. was added 10 equivalents of TFA and 3 hours at room temperature under nitrogen atmosphere. Stirred.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} . The reaction mass was concentrated under reduced pressure. The resulting crude was washed with a saturated NaHCO ₃ solution (300 mL) and then extracted with EtOAc (2 × 200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol. 3.5 g; yield 52% in two steps; confirmed by mass.

ATX-0086: Step 7

To a solution of 4-bromobutyric acid dissolved in DCM (400 mL) and cooled below 0 ° C. was added 1.5 equivalents of EDC. HCl, 2 equivalents of _Et 3 N, and DMAP, in a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution, using an addition funnel, add 20 g of (Z) -non-2-en-1-ol by dissolving in 100 mL of DCM and stirring at room temperature under a nitrogen atmosphere for 24 hours. did.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with water (300 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x150mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (200 mL) and then extracted with EtOAc (150 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / hexane. The alcohol was recovered. 18 g; 55% yield.

ATX-0086: Step 8

  To a solution of 4.0 g of tridecane-7-yl 4-aminobutanoate, 1 equivalent of (Z) -non-2-en-1-yl 4-bromobutanoate in 90 mL of ACN, 1.4 equivalents. Of potassium carbonate was added and the resulting mixture was refluxed at 90 ° C. for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was filtered, washed with ACN (20 mL) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / hexane. The starting amine and bromo compounds were recovered. 2.2 g; yield 30%; confirmed by mass.

ATX-0086: Step 9

  To a solution of 2.2 g tridecane-7-yl (Z) -4-((4- (non-2-en-1-yloxy) -4-oxobutyl) amino) butanoate in 25 mL dry DCM was added Equivalent amounts of triethylamine and triphosgene were added at 0 ° C. at 5 minute intervals under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, 3.5 equivalents of 2- (dimethylamino) propane-1-thiol was added to 7 equivalents of sodium hydride dissolved in dry THF (100 mL) in a two-necked 100 mL round bottom flask stirred at 0 ° C. The hydrochloride was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To the resulting solution, the carbamoyl chloride, dissolved in THF (100 mL), was slowly added via syringe over about 10 minutes. The resulting solution was stirred overnight at 0 ° C. to room temperature under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL) and then EtOAc (150 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x40mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

Initial purification was performed using silica gel (60-120 mesh), the crude compound was adsorbed on 60 g silica gel and poured onto 500 g silica gel in a column. Compound was eluted with 35% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. The crude compound was adsorbed on 18 g of neutral alumina and the resultant was poured on 130 g of neutral alumina placed in a column. Compound eluted with 10% EtOAc / hexane. 1.2 g; yield 43%; confirmed by ¹ H NMR, HPLC, and mass.

ATX-0086 / RL-48A: ¹ H-NMR (PPM, 500 MHz, CDCl ₃ ): δ = 5.64 (m, 1), 5.51 (m, 10, 4.87 (m, 1), 4) .63 (d, J = 7.0, 2), 3.30 to 3.44 (4), 3.02 (t, J = 7.0, 2), 2.52 (t, J = 7.0, 2). 0, 2), 2.26 to 2.36 (4), 2.27 (s, 6), 2.09 (m, 2), 1.82 to 1.96 (4), 1.46 to 1. .54 (4), 1.21-1.40 (24), 0.84-0.91 (9).
Example 8: Synthesis of ATX-0087

  FIG. 7 shows the synthetic route of ATX-0087 with 9 steps.

ATX-0087: Step 1

  A 2 liter two-necked round bottom flask was charged with 20 g of octanoic acid dissolved in DCM (200 mL) and then 1.5 equivalents of oxalyl chloride was slowly added at 0 ° C. with stirring under a nitrogen atmosphere. The resulting reaction mixture was stirred at room temperature for 2 hours. In a separate 2-liter two-necked round-bottom flask, to 2 equivalents of N, O-dimethylhydroxylamine hydrochloride in DCM (200 mL) was added 3 equivalents of trimethylamine stirred at 0 ° C. using an addition funnel. . After concentration under reduced pressure to the resulting solution, the above acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (150 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (250 mL). The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / hexane). Production amount 20 g; yield 84%.

ATX-0087: Step 2

  To a solution of hexylmagnesium bromide (1.5 eq) in THF (100 mL) stirred in a 1 liter two-necked round bottom flask at 0 ° C. under a nitrogen atmosphere was added 20 g of N-methoxy-N-methyl. Octaneamide (1 equivalent) solution (dissolved in 200 mL of THF) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with a saturated NH ₄ Cl solution (250 mL) and then EtOAc (350 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% EtOAc / hexane). Production amount 25 g; yield 65%.

ATX-0087: Step 3

  To a solution of 25 g of tridecane-7-one (1 equivalent) dissolved in MeOH / THF was added 1.5 equivalents of sodium borohydride at 0 ° C. and the resulting solution was stirred at room temperature for 1 hour.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (150 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. Production amount 22 g; yield 90%.

ATX-0087: Step 4

  To a solution of 50 g of 4-aminobutanoic acid in 350 mL of THF at 0 <0> C was added 490 mL of aqueous 1 N NaOH solution, followed by 140 mL of Boc anhydride sequentially over 15 minutes using an addition funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was quenched with 5% HCl (250 mL) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x150mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. 80 g; yield 81%.

ATX-0087: Step 5

To a solution of 17 g of 4-((tert-butoxycarbonyl) amino) butanoic acid dissolved in DCM (250 mL) and cooled below 0 ° C., 1.3 equivalents of EDC. HCl, _Et 3 _N, and 4-dimethylaminopyridine (DMAP), in a nitrogen atmosphere, were successively added at 10 minute intervals. To the resulting solution was added 1 equivalent of tridecane-7-ol at the same temperature by dissolving in DCM (150 mL) using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5). The reaction mass was quenched with water (150 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then EtOAc (200 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. Production 15 g (crude; required compounds and alcohols).

ATX-0087: Step 6

  To a solution of 15.0 g of pentadecane-8-yl 4-((tert-butoxycarbonyl) amino) butanoate dissolved in 80 mL of DCM was added 10 equivalents of TFA at 0 ° C and 3 hours at room temperature under nitrogen atmosphere. Stirred.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} . The reaction mass was concentrated under reduced pressure. The resulting crude was washed with a saturated NaHCO ₃ solution (300 mL) and then extracted with EtOAc (2 × 200 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol. 7 g of yield; 24% yield in 2 steps; confirmed by mass.

ATX-0087: Step 7

To a solution of 4-bromobutyric acid dissolved in DCM (400 mL) and cooled below 0 ° C. was added 1.5 equivalents of EDC. HCl, 2 equivalents of _Et 3 N, and DMAP, in a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution, using an addition funnel, add 20 g of (Z) -non-2-en-1-ol by dissolving in 100 mL of DCM and stirring at room temperature under a nitrogen atmosphere for 24 hours. did.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with water (300 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x150mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (200 mL) and then extracted with EtOAc (150 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / hexane. The alcohol was recovered. 19 g; 55% yield.

ATX-0087: Step 8

  To a solution of 4.0 g of tridecane-7-yl 4-aminobutanoate, 1 equivalent of (Z) -non-2-en-1-yl 4-bromobutanoate in 90 mL of ACN, 1.4 equivalents. Of potassium carbonate was added and the resulting mixture was refluxed at 90 ° C. for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was filtered, washed with ACN (20 mL) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 15% EtOAc / hexane. The starting amine and bromo compounds were recovered. 2.2 g; yield 30%; confirmed by mass.

ATX-0087: Step 9

  To a solution of 2.2 g tridecane-7-yl (Z) -4-((4- (non-2-en-1-yloxy) -4-oxobutyl) amino) butanoate in 25 mL dry DCM was added Equivalent amounts of triethylamine and triphosgene were added at 0 ° C. at 5 minute intervals under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, 3.5 equivalents of 2- (dimethylamino) propane-1-thiol was added to 7 equivalents of sodium hydride dissolved in dry THF (100 mL) in a two-necked 100 mL round bottom flask stirred at 0 ° C. The hydrochloride was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To the resulting solution, the carbamoyl chloride, dissolved in THF (100 mL), was slowly added via syringe over about 10 minutes. The resulting solution was stirred overnight at 0 ° C. to room temperature under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with a saturated NH ₄ Cl solution (75 mL) and then EtOAc (150 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x40mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

Initial purification was performed using silica gel (60-120 mesh), the crude compound was adsorbed on 60 g silica gel and poured onto 500 g silica gel in a column. Compound was eluted with 35% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. The crude compound was adsorbed on 18 g of neutral alumina and the resultant was poured on 130 g of neutral alumina placed in a column. Compound eluted with 10% EtOAc / hexane. 1.2 g; yield 43%; confirmed by ¹ H NMR, HPLC, and mass.

ATX-0087 / RL-48C: ¹ H-NMR (PPM, 500 MHz, CDCl ₃ ): δ = 5.64 (m, 1), 5.52 (m, 1), 4.87 (m, 1), 4.63 (d, J = 7.0, 2), 3.30 to 3.44 (4), 3.02 (t, J = 7.0, 2), 2.52 (t, J = 7) 2.0, 2.26), 2.26 to 2.36 (4), 2.27 (s, 6), 2.09 (m, 2), 1.83 to 1.96 (4), 1.46 to 1.54 (4), 1.21-1.40 (32), 0.85-0.90 (9).
Example 9: Synthesis of ATX-0088

  FIG. 8 shows the synthesis route for ATX-0088, which is described further below.

ATX-0088: Step 1

During two-necked round bottom flask 500mL under N ₂ atmosphere, placed in 25g dissolved in DCM and 200 mL 8- bromooctanoate (1 eq), followed by stirring under a nitrogen atmosphere, oxalyl chloride at 0 ℃ (1.5 equivalents). The resulting reaction mixture was stirred at room temperature for 2 hours.

  In a separate 1 liter two-neck round bottom flask, 2 equivalents of N, O-dimethylhydroxylamine hydrochloride in 300 mL of DCM were added with 3 equivalents of trimethylamine and stirred at 0 ° C. After concentration under reduced pressure to the resulting solution, the acid chloride was added dropwise by dissolving in 500 mL of DCM over 15 minutes using an addition funnel. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (300 mL). The organic layer was separated and the aqueous layer was washed with DCM (2x100mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 10% EtOAc / hexane). 21 g; yield 66%.

ATX-0088: Step 2

  To a solution of 1.3 equivalents of octylmagnesium bromide in THF (100 mL), stirred at 0 ° C. under a nitrogen atmosphere, was added 20 g of N-methoxy-N-methyloctaneamide in 100 mL of THF, which resulted. The reaction mixture was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.7). The reaction mass was quenched with a saturated NH ₄ Cl solution (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 2% ethyl acetate / hexane). Yield was 17.4 g; 68%.

ATX-0088: Step 3

  To a solution of 17 g of hexadecane-7-one (1 eq) dissolved in 135 mL of MeOH / THF was added 1.5 eq of sodium borohydride at 0 ° C and the resulting solution was stirred at room temperature for 1 hour. .

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (50 mL). The methanol was reduced under reduced pressure. The resulting crude was partitioned between EtOAc (200 mL) and water. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 80 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. 14.5 g produced; 85% yield.

ATX-0088: Step 4

  To a solution of 50 g of 4-aminobutanoic acid in 350 mL of THF at 0 <0> C was added 490 mL of aqueous 1 N NaOH solution, followed by 140 mL of Boc anhydride using a funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.5). The reaction mass was quenched with 5% HCl (100 mL) and then EtOAc (200 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. 80 g; yield 81%.

ATX-0088: Step 5

To a solution of one equivalent of 4-((tert-butoxycarbonyl) amino) butanoic acid dissolved in DCM (200 mL) and cooled below 0 ° C. was added three equivalents of EDC. HCl, Et ₃ N (3 eq) and DMAP (0.1 eq) were added sequentially at 10 minute intervals. To the resulting solution was added an alcohol by dissolving in DCM using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mass was quenched with water (100 mL) and the organic layer was separated. The aqueous layer was washed with DCM (2x50mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution and EtOAc (100 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. Generated amount 19 g (crude).

ATX-0088: Step 6

  To a solution of 19 g of hexadecane-7-yl 4-((tert-butoxycarbonyl) amino) butanoate (1 eq.) Dissolved in 140 mL of DCM was added 10 eq. Of TFA at 0 ° C. and at room temperature under nitrogen atmosphere at room temperature. Stir for 3 hours.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.3). The reaction mass was concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (100 mL) and EtOAc (100 mL) was added. The organic layer was separated and concentrated under reduced pressure.

The crude compounds; subjected to column chromatography using (60-120 mesh silica gel 4% MeOH / CHCl _3), to recover the alcohol. 9.4 g produced; 50% yield in two steps; confirmed by mass.

ATX-0088: Step 7

A solution of 30 g of 4-bromobutyric acid (1 equivalent) dissolved in DCM (500 mL) and cooled to 0 ° C. was added with 1.5 equivalents of EDC. HCl, 3 eq. Et ₃ N, and 0.1 eq. DMAP were added sequentially at 10 minute intervals. To this resulting solution is added, using an addition funnel, 0.7 equivalents of (Z) -non-2-en-1-ol by dissolving in 100 mL of DCM, and under a nitrogen atmosphere at room temperature. Stirred for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.7). The reaction mass was quenched with water (100 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution and EtOAc (150 mL) was added. The organic layer was separated and concentrated under reduced pressure.

The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 5% EtOAc / hexane). 27 g; yield 51%; confirmed by ¹ H NMR.

ATX-0088: Step 8

  To a solution of 6 g of hexadecane-8-yl 4-aminobutanoate (1 equivalent) in ACN (70 mL), 1 equivalent of 5 (Z) -non-2-en-1-yl 4-bromobutanoate, 1.2 equivalents of potassium carbonate were added and the resulting mixture was refluxed at 90 ° C. for 3 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.5). The reaction mass was filtered and the filtrate was concentrated under reduced pressure.

  The crude compound was subjected to column chromatography using (100-200 mesh silica gel; 15% EtOAc / hexane). 4.5 g; yield 44%; confirmed by mass.

ATX-0088: Step 9

  4.4 g of (Z) -non-2-en-1-yl 4-((4-oxo-4- (tetradecane-7-yloxy) butyl) amino) butanoate (1 equivalent) dissolved in 30 mL of dry DCM To a solution of was added 0.83 mL of trimethylamine (3 eq) and 418 mg of triphosgene (0.5 eq) at 0 ° C. under a nitrogen atmosphere at 5 min intervals. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  To 192 mg of sodium hydride (10 eq) dissolved in dry THF (25 mL) in a two-neck 100 mL round bottom flask was added 564 mg of 2- (dimethylamino) propane-1-thiol hydrochloride (5 eq) at 0 ° C. The addition was continued with stirring for 5 minutes under a nitrogen atmosphere. To the resulting solution, the carbamoyl chloride, dissolved in THF (35 mL), was added slowly via syringe over about 10 minutes. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 4 hours.

The progress of the reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with a saturated NH ₄ Cl solution (30 mL) and then EtOAc (100 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 50 mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

The first purification was performed using silica gel (60-120 mesh). 5.0 g of the crude compound was adsorbed on 9 g of silica gel and poured onto 90 g of silica gel placed in a column. Compound was eluted with 35% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. 1.5 g of the crude compound was adsorbed on 4 g of neutral alumina, and the resultant was poured on 40 g of neutral alumina placed in a column. Compound eluted with 10% EtOAc / hexane. 1.2 g; yield 21%; confirmed by ¹ H NMR; HPLC; mass.

ATX-0088 / RL-48D: ¹ H-NMR (PPM, 500 MHz, CDCl ₃ ): δ = 5.64 (m, 1), 5.51 (m, 1), 4.87 (m, 1), 4.63 (d, J = 7.0, 2), 3.30 to 3.44 (4), 2.90 (t, J = 7.0, 2), 2.46 to 2.55 (6 ), 2.26 to 2.37 (4), 2.09 (m, 2), 1.71 to 1.80 (4), 1.46 to 1.55 (4), 1.21 to 1. 41 (32), 1.01 (t, J = 7.0, 6), 00.85 to 0.91 (9).
Example 10: Synthesis of ATX-0083

  FIG. 9 shows the synthesis route for ATX-0083, which is described further below.

ATX-0083: Step 1

  In a 500 mL single neck round bottom flask was placed 50 g of octanoic acid (1 eq.) Dissolved in DCM (200 mL) and then 44.6 mL at 0 ° C. via an addition funnel with stirring under a nitrogen atmosphere. Of oxalyl chloride (1.5 eq.) Was slowly added, followed by 1 mL of DMF (catalyst). The resulting reaction mixture was stirred at room temperature for 2 hours.

  In a separate 2-liter two-necked round-bottomed flask, add 144 mL of N, O-dimethylhydroxylamine hydrochloride (2 eq) in DCM (300 mL) stirred at 0 ° C. using an addition funnel. Triethylamine (3 equivalents) was added. After concentration under reduced pressure to the resulting solution, the acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (350 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was diluted with water (300 mL). The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 10% EtOAc / hexane. Amount produced: 55.0 g; yield: 84%

ATX-0083: Step 2

  89.6 g of N-methoxy dissolved in 400 mL of dry ether in a solution of 55 g of heptylmagnesium bromide (1 eq.) In ether, placed in a 2 l two-necked round bottom flask and stirred at 0 ° C. under a nitrogen atmosphere. -N-Methyloctaneamide solution (1.5 eq) was added and the resulting reaction solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7; PMA charring). The reaction mass was quenched with a saturated NH ₄ Cl solution (250 mL). The organic layer was separated and the aqueous layer was washed with ether (2 × 100 mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 2% EtOAc / hexane. Production amount 50.0 g; yield 75%.

ATX-0083: Step 3

  To a solution of 50 g of pentadecan-8-one (1 equiv) dissolved in 290 mL of MeOH / THF at 0 ° C. was added 12.5 g of sodium borohydride (1.5 equiv) and the resulting solution was allowed to cool to room temperature. For 2 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5; PMA charring). The reaction mass was quenched with a saturated NH ₄ Cl solution (80 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (250 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (3x80mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ concentrated under reduced pressure and dried under vacuum to give a white solid. 46.0 g produced; 90% yield.

ATX-0083: Step 4

  To a solution of 50 g of 4-aminobutanoic acid (1 eq.) Dissolved in THF at 0 ° C. was added 490 mL of 1N aqueous NaOH solution (1 eq.) Followed by 140 mL of Boc anhydride (1.3 eq.) Over 15 min. Were added sequentially using an addition funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was quenched with 5% HCl (350 mL) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x150mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to give a gummy liquid. 77.0 g produced; 78% yield.

ATX-0083: Step 5

The synthesis was performed in four times. In each case, a solution of 23 g of 4-((tert-butoxycarbonyl) amino) butanoic acid (1 equivalent) in DCM (400 mL) cooled below 0 ° C. was added with 32.3 g of EDC. HCl (1.5 eq), 47 mL of _Et 3 N (3 eq), and 1.3g of DMAP (0.1 eq), under a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution was added, using an addition funnel, 20 g of pentadecane-8-ol (0.77 equiv) by dissolving in DCM (200 mL) and stirring at room temperature under a nitrogen atmosphere for 24 hours. .

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.4). The reaction mass was quenched with water (250 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. To the resulting crude was washed with a saturated NaHCO ₃ solution (150 mL) and EtOAc (250 mL) was added. The organic layer was separated, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure, then carried on crude to the next step. Produced amount 105g (crude; necessary compound and alcohol)

ATX-0083: Step 6

  To a solution of 105 g of pentadecane-8-yl 4-((tert-butoxycarbonyl) amino) butanoate (1 eq) dissolved in 450 mL of DCM at 0 ° C. was added 194 mL of TFA (10 eq) and under a nitrogen atmosphere. And stirred at room temperature for 3 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} .

The reaction mass was concentrated under reduced pressure. The resulting crude was stirred with saturated NaHCO ₃ solution (200 mL) for 10 minutes, then with EtOAc (300 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

The crude compound was subjected to column chromatography (silica gel 60-120 mesh) using 4% MeOH / CHCl ₃ and 1 mL of triethylamine. 60.0 g produced in two steps; yield 54%.

ATX-0083: Step 7

The reaction was performed in two portions, in each case 29.3 g of EDC. Was dissolved in a solution of 20 g of 6-bromohexanoic acid (1 eq.) Dissolved in DCM (300 mL) and cooled below 0 ° C. HCl (1.5 eq), 42.8 ml of _Et 3 N (3 eq), and 1.2g of DMAP (0.1 eq) under a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution was added (by dissolving in 100 mL of DCM) 14.5 g of (Z) -non-2-en-1-ol (1 equivalent) using an addition funnel and a nitrogen atmosphere The mixture was stirred at room temperature for 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with water (200 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then extracted with EtOAc (2 × 150 mL). The organic layer was separated, dried over anhydrous Na ₂ SO _4, and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 4% EtOAc / hexane. The alcohol reactant was collected. 36.0 g produced; 55% yield.

ATX-0083: Step 8

  The reaction was performed in six parts. In each case, 10 g of pentadecan-8-yl 4-aminobutanoate (Int 6, 1 eq.), 10.1 g of (Z) -non-2-en-1-yl 6-bromohexanoate in 120 mL of ACN To a solution of (Int 7, 1 eq) was added 6.1 g of anhydrous potassium carbonate (1.4 eq) and the resulting mixture was refluxed at 90 ° C for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was filtered, washed with ACN (2 × 20 mL) and the filtrate was concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (silica gel 100-200 mesh) using 20-80% EtOAc / hexane. Starting material was collected. 36.9 g produced; 35% yield.

ATX-0083: Step 9

  The reaction was performed in three parts. In each case, 10 g of (Z) -non-2-en-1-yl 6-((4-oxo-4- (pentadecane-8-yloxy) butyl) amino) hexanoate (1 equivalent) dissolved in 100 mL of dry DCM )), 7.5 mL of triethylamine (3 eq.) And 2.68 g of triphosgene (0.5 eq.) Were added at 0 ° C. at 5 minute intervals under a nitrogen atmosphere. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, in a two-necked 500 mL RB flask, stirred at 0 ° C., was added 8.9 g of 2- (dimethylamino) ethane in a suspension of 3 g of sodium hydride (7 equivalents) in dry THF (100 mL). -1-thiol hydrochloride (3.5 eq) was added and stirring was continued for 5 minutes under a nitrogen atmosphere. To the resulting solution, the carbamoyl chloride, dissolved in dry THF (200 mL), was added slowly via syringe over about 10 minutes. The resulting solution was stirred overnight at room temperature under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with a saturated NH ₄ Cl solution (100 mL) and then EtOAc (350 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 80 mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

  The first purification was performed using neutral alumina. The crude compound dissolved in hexane was packed on top of neutral alumina (700 g packed in column). Compound eluted with 8-10% EtOAc / hexane. The second purification was performed using silica gel (100-200 mesh). The compound dissolved in hexane was packed on top of the silica gel (500 g packed in a column). Compound eluted with 20-25% EtOAc / hexane. The final compound (dissolved in hexane) was subjected to charcoal treatment (200 mg / g), filtered through a celite bed (after stirring for 20 minutes), and then passed through a syringe end membrane filter (PTFE; 0.2 micron, 25 mm diameter). . The obtained filtrate was concentrated under reduced pressure. Production amount: 15.5 g; yield: 41%.

ATX-0083 / RL-47B: ¹ H-NMR (PPM, 500 MHz, CDCl ₃ ): δ = 5.64 (m, 1), 5.52 (m, 1), 4.87 (m, 1), 4.62 (d, J = 7.0, 2), 3.24-3.42 (4), 3.02 (t, J = 7.0, 2), 2.53 (t, J = 7) 2.0, 2), 2.26 to 2.34 (4), 2.26 (s, 6), 2.10 (m, 2), 1.45 to 1.70 (6), 1.20 to 20. 1.41 (34), 0.84-0.92 (9).
Example 11: Synthesis of ATX-0084

  FIG. 10 shows the synthesis route for ATX-0084, which is described further below.

ATX-0084: Step 1

  In a 500 mL single neck round bottom flask was placed 30 g of heptanoic acid (1 eq.) Dissolved in DCM (200 mL), then 26.7 g of oxalyl chloride (1. 5 eq.) Was slowly added, followed by 1 mL of DMF (catalyst). The resulting reaction mixture was stirred at room temperature for 2 hours.

  In a separate 1 l two-necked round bottom flask, 86.6 mL of 40.5 g of N, O-dimethylhydroxylamine hydrochloride (2 eq) in DCM (250 mL) was stirred at 0 ° C. using an addition funnel. Of trimethylamine (3 equivalents) was added. After concentration under reduced pressure to the resulting solution, the acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (100 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (250 mL). The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 silica gel) using 10% EtOAc / hexane. 38.0 g produced; 84% yield.

ATX-0084: Step 2

  2.3 g dissolved in 250 mL ether in a solution of 8 g hexylmagnesium bromide (1 eq) in 250 mL dry ether, placed in a 1 liter two-necked round bottom flask and stirred at 0 ° C. under a nitrogen atmosphere. N-Methoxy-N-methylheptaneamide (0.5 eq) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with saturated NH ₄ Cl solution (200 mL). The organic layer was separated and the aqueous layer was washed with ether (2 × 100 mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 2% EtOAc / hexane. 30.8 g produced; 71% yield.

ATX-0084: Step 3

  To a solution of 30 g of tridecane-7-one (1 eq) dissolved in 200 mL of MeOH / THF at 0 ° C. was added 8.5 g of sodium borohydride (0.5 eq) and the resulting solution was allowed to cool to room temperature. For 2 hours.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mass was quenched with a saturated NH ₄ Cl solution (80 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (200 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 70 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. 27.2 g produced; 90% yield.

ATX-0084: Step 4

  To a solution of 5 g of 6-aminohexanoic acid (1 equiv.) Dissolved in 120 mL of THF at 0 ° C. was added 125 mL of 1N aqueous NaOH solution followed by 34 mL of Boc anhydride (1.3 equiv.) Over 15 min. They were added sequentially using an addition funnel. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was quenched with 5% HCl (100 mL) and then EtOAc (150 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. 22.4 g produced; 85% yield.

ATX-0084: Step 5

A solution of 10 g of 6-((tert-butoxycarbonyl) amino) hexanoic acid (1 equivalent) dissolved in DCM (200 mL) and cooled below 0 ° C. was charged with 10.7 g of EDC. HCl (1.3 eq), 18 mL of _Et 3 N (3 eq), and 525mg of DMAP (0.1 eq) under a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution is added 6 g of tridecane-7-ol (Int3, 0.7 equiv.) At the same temperature by dissolving in DCM (50 mL) using an addition funnel and under a nitrogen atmosphere at room temperature. For 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.4). The reaction mass was quenched with water (150 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x75mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (100 mL), then extracted and added with EtOAc (2 × 100 mL). The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. 8.5 g (crude; required compounds and alcohols) produced.

ATX-0084: Step 6

  To a solution of 10 g of tridecane-7-yl 6-((tert-butoxycarbonyl) amino) hexanoate (1 eq.) Dissolved in 65 mL of DCM at 0 ° C. was added 18.5 mL of TFA (10 eq.) And added nitrogen. The mixture was stirred at room temperature for 3 hours under an atmosphere.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} . The reaction mass was concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (100 mL) and then extracted with EtOAc (3 × 100 mL). The organic layer was separated and concentrated under reduced pressure.

The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol starting material. 4.5 g in 2 steps; yield 33%.

ATX-0084: Step 7

A solution of 20 g of 6-bromohexanoic acid (1 equivalent) dissolved in DCM (300 mL) and cooled below 0 ° C. was charged with 29.3 g of EDC. HCl (1.5 eq), 42.8 ml of _Et 3 N (3 eq), and 1.2g of DMAP (0.1 eq) under a nitrogen atmosphere, were successively added at 10 minute intervals. To the resulting solution was added 14.5 g of (Z) -non-2-en-1-ol (1 eq.) Dissolved in 100 mL of DCM using an addition funnel and under a nitrogen atmosphere at room temperature. For 24 hours.

The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7). The reaction mass was quenched with water (200 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then extracted with EtOAc (2 × 150 mL). The organic layer was separated, dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 4% EtOAc / hexane. The alcohol starting material was recovered. 18.0 g produced; 55% yield.

ATX-0084: Step 8

  4.5 g of tridecane-7-yl 6-aminohexanoate (Int6, 1 equivalent) in 90 mL of ACN, 4.5 g of (Z) -non-2-en-1-yl 6-bromohexanoate ( To a solution of Int 7, 1 equivalent) was added 2.7 g of potassium carbonate (1.4 equivalents) and the resulting mixture was refluxed at 90 ° C for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} . The reaction mass was filtered, washed with ACN (2 × 20 mL) and the filtrate was concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 20% EtOAc / hexane. Starting material was collected. 3.0 g; yield 37%.

ATX-0084: Step 9

  2.5 g of (Z) -non-2-en-1-yl 6-((6-oxo-6- (tridec-7-yloxy) hexyl) amino) hexanoate (1 equivalent) dissolved in 30 mL of dry DCM To a solution of was added 1.8 mL of triethylamine (3 eq.) And 672 mg of triphosgene (0.5 eq.) Under a nitrogen atmosphere at 0 ° C. at 5 minute intervals. The resulting solution was stirred at room temperature under a nitrogen atmosphere for 1 hour. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, a suspension of 761 mg of sodium hydride in dry THF (50 mL) was added to 2.2 g of 2- (dimethylamino) ethane-1- in a two-neck 250 mL round bottom flask stirred at 0 ° C. Thiol hydrochloride (3.5 eq) was added and stirring continued under a nitrogen atmosphere for 5 minutes. To the resulting solution, the carbamoyl chloride, dissolved in THF (60 mL), was added slowly via syringe over about 10 minutes. The resulting solution was stirred overnight at room temperature under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5; PMA carbonization). The reaction mass was quenched with a saturated NH ₄ Cl solution (60 mL) and then EtOAc (130 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x40mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography.

  The first purification was performed using silica gel (100-200 mesh). 4.6 g of the crude compound was adsorbed on 10.0 g of silica gel and poured onto 90.0 g of silica gel placed in a column. Compound was eluted with 50% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. 2.0 g of the crude compound was adsorbed on 6.0 g of neutral alumina and the resulting was poured on 40.0 g of neutral alumina placed in a column. Compound was eluted with 20% EtOAc / hexane. 1.2 g yield; 38% yield (300 mg mixture).

ATX-0084 / RL-47C: ¹ H-NMR (PPM, 500 MHz, CDCl ₃ ): δ = 5.64 (m, 1), 5.52 (m, 1), 4.86 (m, 1), 4.62 (d, J = 7.0, 2), 3.22 to 3.35 (4), 3.01 (t, J = 7.0, 2), 2.53 (t, J = 7) 2.0, 2), 2.25 to 2.34 (4), 2.27 (s, 6), 2.10 (m, 2), 1.45 to 1-73 (10), 1.20 to 20. 1.40 (30), 00.84-0.91 (9).
Example 12: Synthesis of ATX-0061

  FIG. 11 shows the synthesis route for ATX-0061, which is described further below.

ATX-0061: Step 1

  12 g of glycine ester (1 equivalent) was dissolved in THF (100 mL) and cooled below 0 ° C. To this solution, 24.2 mL of triethylamine (1.5 eq) and 38.11 g of Boc anhydride (1.5 eq) were sequentially added via an addition funnel.

  The progress of the reaction was monitored by TLC using 50% EtOAc / hexane; Rf: 0.4.

  The reaction mass was quenched with water and EtOAc (100 mL) was added after 16 hours. The organic layer was separated, the aqueous layer was washed with EtOAc (2 × 40 mL), and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

  The crude product was applied on 60-120 silica gel (25% EtOAc / hexane). 20.8 yield; 88% yield.

ATX-0061: Step 2

  To a solution of 18.9 g of N-Boc glycine ester (1 eq) dissolved in THF (130 mL) was added an aqueous solution of 5.85 g of LiOH (1.5 eq) and the resulting solution was allowed to stand at room temperature for 4 hours. Stirred.

  The reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.3) and no SM was present.

  The reaction mass was concentrated and the crude mass was quenched with 5% HCl (pH 3), then extracted with EtOAc (4 × 80 mL), dried over sodium sulfate and concentrated under reduced pressure to give the compound. 15 g; yield 92%; confirmed by mass.

ATX-0061: Step 3

To a solution of 5 g of N-Boc-glycine ester (Int1, 1 equiv.) Dissolved in DCM (30 mL) and cooled below 0 ° C., 4.5 mL of Et ₃ N (1.2 equiv.) And 6.44 g. EDC. HCl (1.2 eq) was added. To this reaction solution was added 5.12 g heptaden-9-ol (0.7 eq) in 20 mL DCM and stirred overnight at room temperature.

Starting material was confirmed by TLC to be absent (10% EtOAc / hexane; Rf: 0.6). The reaction mass was diluted with saturated NaHCO ₃ solution, the organic layer was separated, the aqueous layer was washed with DCM (2 × 30 mL), dried over sodium sulfate and concentrated under reduced pressure. The crude (6.8 g; mixture of product and alcohol) was taken to the next step.

ATX-0061: Step 4

  4 g of heptadecan-9-yl (tert-butoxycarbonyl) glycinate (Int2, 1 eq.) Is dissolved in DCM (40 mL), cooled to 0 ° C., 7.4 mL of TFA (10 eq.) Is added and at room temperature Stir for 1 hour.

  Completion of the reaction was confirmed by TLC in 2 hours (10% EtOAc / hexane; Rf: 0.5).

  The reaction mass is concentrated under reduced pressure, the remaining mass is washed with saturated sodium bicarbonate solution (30 mL), extracted with EtOAc (3 × 30 mL), the organic layer is dried over sodium sulfate and concentrated under reduced pressure , Int3.

The crude product was subjected to column chromatography using Et ₃ N of 1~3% MeOH / CHCl ₃ and 2 mL (silica, 60-120). Produced amount 1 g; confirmed by ¹ H-NMR and mass.

ATX-0061: Step 5

To a solution of 4 g of bromoacetic acid (1 eq) dissolved in DCM (35 mL) and cooled below 0 ° C., 4.7 mL of Et ₃ N (1.2 eq) and 354 mg of DMAP (0.1 eq) Followed by the addition of 13.23 g of HATU (1.2 eq). To this reaction solution was added 2.88 g of (Z) -non-2-en-1-ol (0.7 eq) in 20 mL of DCM and stirred at room temperature overnight.

  The reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.7).

The reaction mass was diluted with saturated NaHCO ₃ solution (80 mL), the organic layer was separated, the aqueous layer was washed with DCM (40 mL), dried over sodium sulfate and concentrated under reduced pressure. The residual mass was purified by silica gel (60-120) column chromatography (1.5% EtOAc / hexane). 4 g; yield 52%.

ATX-0061: Step 6

  1 g of heptadecan-9-ylglycinate (Int3, 1 eq.) Was dissolved in THF (25 mL), 0.5 mL of TEA (1.3 eq.) And 1.08 g of (Z) -non-2-ene-1-. An yl 2-bromoacetate derivative (Int4, 1.3 equivalents) was added and stirred at room temperature overnight.

  The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.4). The reaction mixture was diluted with water (30 mL), extracted with EtOAc (20 mL × 2), the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

  The residual mass was purified by column (silica gel; 100-200) chromatography (2% EtOAc / hexane). 700 mg; yield 47%; confirmed by mass.

ATX-0061: Step 7

700 mg of heptadecan-9-yl (Z)-(2- (non-2-en-1-yloxy) -2-oxoethyl) glycinate) (1 equivalent) dissolved in 15 mL of DCM and cooled below 5 ° C. the solution, _Et 3 N (3 eq) of 0.4 mL, followed by 209mg of triphosgene (0.5 eq) was added portionwise over 10 minutes.

  Progress of the reaction mixture, monitored by TLC, was complete in 0.5 h and the reaction mass was concentrated under reduced pressure.

  To a solution of 423 mg of N, N-dimethylethanethiol hydrochloride (3 eq) in dry THF (10 mL) and DMF (3 mL) stirred at 0 ° C. under a nitrogen atmosphere was added 144 mg of sodium hydride (6 eq). Was added. After 10 minutes, the above solution was added to the reaction mass by dissolving the solution in THF (15 mL). The resulting solution was stirred at room temperature for 1 hour.

Completion of the reaction was confirmed by TLC after 1 hour (10% MeOH / CHCl ₃ ; Rf: 0.5).

The reaction mass was quenched with saturated NH ₄ Cl solution (20 mL) and water (20 mL) and EtOAc (30 mL) were added. The aqueous layer was washed with EtOAc (2 × 20 mL) and the combined organic layers were washed with a brine solution (20 mL). The organic layer was dried over _Na 2 SO _4, and concentrated under reduced pressure.

The crude was subjected to column chromatography using silica gel (100-200) with 15% EtOAc / hexane and then using neutral alumina with 15% EtOAc / hexane to give pure compound. Produced amount: 520 mg; yield: 58%; confirmed by ¹ H-NMR, HPLC, and mass.

ATX-0061 / RL-42D: ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 5.67 (m, 1), 5.51 (m, 1), 4.92 (m, 1), 4.70 (m, 2), 4.16 to 4.27 (4), 3.07 (m, 2), 2.53 (m, 2), 2.27 (s, 6), 2.10 (M, 2), 1-47 to 1.57 (4), 1.19 to 1.40 (32), 0.83 to 0.92 (9).
Example 13: Synthesis of ATX-0063

  FIG. 12 shows the synthesis route for ATX-0063, which is described further below.

ATX-0063: Step 1

  12 g of glycine ester (1 equivalent) was dissolved in THF (100 mL) and cooled below 0 ° C. To this solution, 24.2 mL of triethylamine (1.5 eq) and 38.11 g of Boc anhydride (1.5 eq) were sequentially added via an addition funnel.

  The progress of the reaction was monitored by TLC using 50% EtOAc / hexane; Rf: 0.4.

  The reaction mass was quenched with water and EtOAc (100 mL) was added after 16 hours. The organic layer was separated, the aqueous layer was washed with EtOAc (2 × 40 mL), and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

  The crude product was applied on 60-120 silica gel (25% EtOAc / hexane). 20.8 yield; 88% yield.

ATX-0063: Step 2

  To a solution of 18.9 g of N-Boc glycine ester (1 eq) dissolved in THF (130 mL) was added an aqueous solution of 5.85 g of LiOH (1.5 eq) and the resulting solution was allowed to stand at room temperature for 4 hours. Stirred.

  The reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.3) and no starting was present in the reaction product.

  The reaction mass was concentrated and the crude mass was quenched with 5% HCl (pH 3), then extracted with EtOAc (4 × 80 mL), dried over sodium sulfate and concentrated under reduced pressure to give the compound. 15 g; yield 92%; confirmed by mass.

ATX-0063: Step 3

To a solution of 5 g of N-Boc-glycine ester (Int1, 1 equiv.) Dissolved in DCM (50 mL) and cooled below 0 ° C., 4.5 mL of Et ₃ N (1.2 equiv.) And 6.4 g. EDC. HCl (1.2 eq) was added. To this reaction solution was added 3.4 g undecane-6-ol (0.7 eq) in 20 mL DCM and stirred at room temperature overnight.

Starting material was confirmed by TLC to be absent (15% EtOAc / hexane; Rf: 0.6). The reaction mass was diluted with saturated NaHCO ₃ solution (20 mL), the organic layer was separated, the aqueous layer was washed with DCM (2 × 40 mL), dried over sodium sulfate and concentrated under reduced pressure. After column filtration, the crude (5.5 g; mixture of product and alcohol) was carried on to the next step.

ATX-0063: Step 4

  Dissolve 3.3 g of crude undecane-6-yl (tert-butoxycarbonyl) glycinate (Int2, 1 eq.) In DCM (20 mL), cool to 0 ° C. and add 7.6 mL of TFA (10 eq.). And stirred at room temperature for 1 hour.

  Completion of the reaction was confirmed by TLC in 2 hours (10% MeOH / DCM; Rf: 0.5). The reaction mass is concentrated under reduced pressure, the remaining mass is washed with saturated sodium bicarbonate solution (50 mL), extracted with EtOAc (3 × 25 mL), the organic layer is dried over sodium sulfate and concentrated under reduced pressure , Int3.

The crude product was subjected to column chromatography using Et ₃ N of 1~3% MeOH / CHCl ₃ and 2 mL (silica, 60-120). Produced amount 1.2 g; yield 40%; confirmed by ¹ H-NMR and mass.

ATX-0063: Step 5

A solution of 4 g of bromoacetic acid (1 eq.) Dissolved in DCM (35 mL) and cooled below 0 ° C. was added with 4.7 mL of Et ₃ N (1.2 eq.) Followed by 13.23 g of HATU (1 eq.). .2 eq), and 354 mg of DMAP (0.1 eq). To this reaction solution was added 2.88 g of (Z) -non-2-en-1-ol (0.7 eq) in 20 mL of DCM and stirred at room temperature overnight.

  The reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.7).

The reaction mass was diluted with saturated NaHCO ₃ solution (80 mL), the organic layer was separated, the aqueous layer was washed with DCM (40 mL), dried over sodium sulfate and concentrated under reduced pressure. The residual mass was purified by silica gel (60-120) column chromatography (1.5% EtOAc / hexane). 4 g; yield 52%.

ATX-0063: Step 6

  Dissolve 1.2 g of undecane-6-ylglycinate (Int3, 1 eq.) In 25 mL of THF, 0.9 mL of TEA (1.3 eq.) And 1.37 g of (Z) -non-2-ene-1. -Yl 2-bromoacetate (Int4, 1 eq) was added and stirred at room temperature overnight.

  The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mixture was diluted with water (30 mL), extracted with EtOAc (20 mL × 2), the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

  The residual mass was purified by column (silica gel; 100-200) chromatography (3% EtOAc / hexane). 800 mg; yield 37%; confirmed by mass.

ATX-0063: Step 7

Of 800 mg of (Z) -non-2-en-1-yl (2-oxo-2- (undecane-6-yloxy) ethyl) glycinate (1 equivalent) dissolved in DCM and cooled below 5 ° C. solution, _Et 3 N (3 eq) of 0.4 mL, followed by 209mg of triphosgene (0.5 eq) was added portionwise over 10 minutes.

  Progress of the reaction mixture, monitored by TLC, was complete in 1 hour and the reaction mass was concentrated under reduced pressure.

  To a solution of 423 mg of N, N-dimethylethanethiol hydrochloride (3 eq) in dry THF and DMF (10 mL and 5 mL, respectively) stirred at 0 ° C. under a nitrogen atmosphere, 144 mg of sodium hydride (6 eq) Was added. After 10 minutes, the above solution was added to the reaction mass by dissolving in THF. The resulting solution was stirred at room temperature for 1 hour.

Completion of the reaction was confirmed by TLC after 1 hour (70% EtOAc / hexane; Rf: 0.4). The reaction mass was quenched with saturated NH ₄ Cl solution (25 mL) and water (20 mL) and EtOAc (20 mL) were added. The aqueous layer was washed with EtOAc (2 × 20 mL) and the combined organic layers were washed with a brine solution (20 mL). The organic layer was dried over _Na 2 SO _4, and concentrated under reduced pressure.

The crude was subjected to column chromatography using silica gel (100-200) with 20% EtOAc / hexane and then using neutral alumina with 5% EtOAc / hexane to give pure compound. Production amount 510 mg; yield 48%; confirmed by ¹ H-NMR, HPLC, and mass.

ATX-0063 / RL-42A: ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 5.67 (m, 1), 5.52 (m, 1), 4.92 (m, 1), 4.70 (m, 2), 4.15 to 4.27 (4), 3.06 (m, 2), 2.53 (m, 2), 2.27 (s, 6), 2.09 (M, 2), 1.47 to 1.57 (4), 1.20 to 1.41 (20), 0.82 to 0.92 (9).
Example 14: Synthesis of ATX-0064

  FIG. 13 shows the synthesis route for ATX-0064, which is described further below.

ATX-0064: Step 1

  12 g of ethyl glycinate (1 eq) was dissolved in THF (100 mL) and cooled below 0 ° C. To this resulting solution, 24.2 mL of triethylamine (1.5 eq) and 38.11 g of Boc anhydride (1.5 eq) were sequentially added via an addition funnel.

  The progress of the reaction was monitored by TLC using 50% EtOAc / hexane; Rf: 0.4.

  The reaction mass was quenched with water and EtOAc (100 mL) was added after 16 hours. The organic layer was separated, the aqueous layer was washed with EtOAc (2 × 40 mL), and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

  The crude product was applied on 60-120 silica gel (25% EtOAc / hexane). 20.8 yield; 88% yield.

ATX-0064: Step 2

  To a solution of 18.9 g of N-Boc glycine ester (1 eq) dissolved in THF (130 mL) was added an aqueous solution of 5.85 g of LiOH (1.5 eq) and the resulting solution was allowed to stand at room temperature for 4 hours. Stirred.

  The reaction was monitored by TLC (60% EtOAc / hexane; Rf: 0.3) and no starting material was present in the reaction product.

  The reaction mass was concentrated and the crude mass was quenched with 5% HCl (pH 3), then extracted with EtOAc (4 × 80 mL), dried over sodium sulfate and concentrated under reduced pressure to give the compound. 15 g; yield 92%; confirmed by mass.

ATX-0064: Step 3

To a solution of 5 g of N-Boc-glycine ester (Int1, 1 eq) dissolved in DCM (50 mL) and cooled below 0 ° C., 4.5 mL of Et ₃ N (1.2 eq) and 6.4 g EDC. HCl (1.2 eq) was added. To this reaction solution was added 4.84 g of hexadecane-10-ol (0.7 eq) in 15 mL of DCM and stirred overnight at room temperature.

Starting material was confirmed by TLC to be absent (15% EtOAc / hexane; Rf: 0.6). The reaction mass was diluted with saturated NaHCO ₃ solution, the organic layer was separated, the aqueous layer was washed with DCM (2 × 30 mL), dried over sodium sulfate and concentrated under reduced pressure.

  After column filtration, the crude (5.5 g; mixture of product and alcohol) was carried on to the next step.

ATX-0064: Step 4

  Dissolve 3.85 g of crude heptadecan-9-yl (tert-butoxycarbonyl) glycinate (Int2, 1 eq.) In 30 mL of DCM, cool to 0 ° C. and add 7.4 mL of TFA (10 eq.) Stirred at room temperature for 1 hour.

  Completion of the reaction was confirmed by TLC in 2 hours (10% MeOH / DCM; Rf: 0.5).

  The reaction mass is concentrated under reduced pressure, the remaining mass is washed with saturated sodium bicarbonate solution (30 mL), extracted with EtOAc (3 × 30 mL), the organic layer is dried over sodium sulfate and concentrated under reduced pressure , Int3.

The crude product was subjected to column chromatography using Et ₃ N of 1~3% MeOH / CHCl ₃ and 2 mL (silica, 60-120). Produced amount 2.2 g; confirmed by ¹ H-NMR and mass.

ATX-0064: Step 5

A solution of 4 g of bromoacetic acid (1 eq.) Dissolved in DCM (35 mL) and cooled below 0 ° C. was added with 4.7 mL of Et ₃ N (1.2 eq.) Followed by 13.23 g of HATU (1 eq.). .2 eq), and 354 mg of DMAP (0.1 eq). To this reaction solution was added 2.88 g of (Z) -non-2-en-1-ol (0.7 eq) in 20 mL of DCM and stirred at room temperature overnight.

  The reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.7).

The reaction mass was diluted with saturated NaHCO ₃ solution (80 mL), the organic layer was separated, the aqueous layer was washed with DCM (40 mL), dried over sodium sulfate and concentrated under reduced pressure.

  The residual mass was purified by silica gel (60-120) column chromatography (1.5% EtOAc / hexane). 4 g; yield 52%.

ATX-0064: Step 6

  2.1 g of hexadecane-8-ylglycinate (Int3, 1 eq) was dissolved in 50 mL of THF, 1.2 mL of TEA (1.3 eq) and 2.39 g of (Z) -non-2-ene-1. -Yl 2-bromoacetate (Int4, 1.3 eq) was added and stirred at room temperature overnight.

  The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5). The reaction mixture was diluted with water (30 mL), extracted with EtOAc (2 × 30 mL), the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure.

  The residual mass was purified by column (silica gel; 100-200) chromatography (3% EtOAc / hexane). 2.2 g; yield 65%; confirmed by mass.

ATX-0064: Step 7

2.2 g of heptadecan-9-yl (Z)-(2- (non-2-en-1-yloxy) -2-oxoethyl) glycinate) (1) dissolved in 15 mL of DCM and cooled below 5 ° C. equiv), _Et 3 N (3 equiv of 1.6 mL), followed by 678mg of triphosgene (0.5 eq) was added portionwise over 10 minutes.

  Progress of the reaction mixture, monitored by TLC, was complete in 1 hour and the reaction mass was concentrated under reduced pressure.

  To a solution of 3.94 g of N, N-dimethylethanethiol hydrochloride (7 equivalents) in dry THF and DMF (35 mL and 15 mL, respectively) stirred at 0 ° C. under a nitrogen atmosphere was added 672 mg of sodium hydride (7 Eq.) Was added. After 10 minutes, the above solution was added to the reaction mass by dissolving in THF. The resulting solution was stirred at room temperature for 1 hour.

Completion of the reaction was confirmed by TLC after 1 hour (70% EtOAc / hexane; Rf: 0.4). The reaction mass was quenched with a saturated NH ₄ Cl solution (25 mL) and water (20 mL) and EtOAc (20 mL) were added. The aqueous layer was washed with EtOAc (2 × 20 mL) and the combined organic layers were washed with a brine solution (20 mL). The organic layer was dried over _Na 2 SO _4, and concentrated under reduced pressure.

The crude was subjected to column chromatography using silica gel (100-200) with 25% EtOAc / hexane and then neutral alumina with 15-20% EtOAc / hexane to give pure compound. 1.0 mg; yield 40%; confirmed by ¹ H-NMR, HPLC, and mass.

ATX-0064 / RL-42C: ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 5.67 (m, 1), 5.50 (m, 1), 4.92 (m, 1), 4.70 (t, J = 7.0, 2), 3.06 (, m, 2), 2.53 (m, 2), 2.27 (s, 6), 1.47 to 1.57 (4), 1.17 to 1.40 (30), 0.82 to 0.93 (9).
Example 15: Synthesis of ATX-0081

  FIG. 14 shows the synthesis route for ATX-0081, which is described further below.

ATX-0081: Step 1

  To a solution of heptylmagnesium bromide (1.5 eq.) In 370 mL of dry THF (formed in situ) cooled to -15 ° C under a nitrogen atmosphere, via an addition funnel, ethylformate dissolved in 80 mL of THF. Mate (0.5 eq) was added dropwise over 20 minutes and the resulting reaction mixture was warmed to room temperature and stirred overnight.

  The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5).

The reaction mass was quenched with saturated NH ₄ Cl solution (500 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography on silica gel (60-120 mesh) using 20% EtOAc / hexane. Production amount: 169.0 g; yield: 60%.

ATX-0081: Step 2

  To a solution of 100 g of 4-aminobutanoic acid (1 eq) in THF was added 1 N aqueous NaOH solution (1.065 lit) (1.1 eq) followed by 1.3 eq of Boc anhydride in an ice bath. It was added sequentially using an addition funnel over a 15 minute period. The resulting solution was warmed to room temperature and stirred for 4 hours.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.5).

  The reaction mass was quenched with 5% HCl (1 L) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 200 mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid.

  The crude compound was subjected to column chromatography on silica gel (60-120 mesh) using 80-100% EtOAc / hexane. Production amount: 161.0 g; yield: 82%.

ATX-0081: Step 3

To a solution of 3 × 17.8 g of 4-((tert-butoxycarbonyl) amino) butanoic acid (1 eq) in DCM (350 mL) cooled in an ice bath, 2 × 25.1 g of EDC. HCl (1.5 _eq), Et 3 N (2.0 eq), and 3 × 1.0 g of DMAP (0.1 eq), under a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution, 3 × 20.0 g of alcohol (1.0 eq) (in 150 mL of DCM) is added dropwise at the same temperature via an addition funnel and the resulting reaction mass is brought to room temperature. Warm and stir under nitrogen for 24 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5).

The reaction mass was quenched with water (450 mL) and the organic layer was separated. The aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was stirred with saturated NaHCO ₃ solution (300 mL) for 5 minutes and the aqueous phase was extracted with EtOAc (3 × 150 mL). The organic layer was separated, dried over anhydrous Na ₂ SO ₄ , concentrated under reduced pressure and carried on crude to the next step. 69.0 g of product (crude; necessary compounds and alcohol);

ATX-0081: Step 4

  To a solution of 69.0 g of pentadecane-8-yl 4-((tert-butoxycarbonyl) amino) butanoate (1 eq) dissolved in DCM was added TFA (10 eq) in an ice bath and the resulting reaction was performed. The solution was warmed to room temperature and stirred under a nitrogen atmosphere for 3 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} . The reaction mass was concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (450 mL) and then extracted with EtOAc (3 × 150 mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol. 47.0 g produced; 57% yield in two steps;

ATX-0081: Step 5

A solution of 50.0 g of 4-bromobutyric acid (1.0 eq) in DCM (450 mL), placed in a 2 L RB flask and cooled below 0 ° C., was charged with 86.0 g of EDC. HCl (1.5 eq), 83.3 ml of a _Et 3 N (2.0 eq), and 3.6g of DMAP (0.1 eq), under a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution, 42.5 g of alcohol (1.0 eq) (by dissolving in 200 mL of DCM) is added using an addition funnel and the resulting solution is warmed to room temperature and placed under a nitrogen atmosphere. For 24 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7).

The reaction mass was quenched with water (250 mL) and the organic layer was separated. The aqueous layer was washed with DCM (2x150mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (150 mL) and then extracted with EtOAc (2 × 150 mL). The organic layer was separated and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography on silica gel (60-120 mesh silica gel) using 5% EtOAc / hexane. Production amount 55.0 g; yield 63%

ATX-0081: Step 6

  2 × 20.0 g of pentadecane-8-yl 4-aminobutanoate (1 equivalent) in 180 mL of ACN, 2 × 18.5 g of (Z) -non-2-en-1-yl 4-bromobuta To the solution of noate (1 equivalent) was added 2 × 17.6 g of potassium carbonate (2 equivalents) and the resulting mixture was refluxed at 70 ° C. for 5 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.4). The reaction mass was filtered, washed with ACN (2 × 20 mL) and the filtrate was concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 30% EtOAc / hexane. SM (amine) was recovered. Production amount 24.0 g; yield 36%.

ATX-0081: Step 7

  24.0 g of (Z) -non-2-en-1-yl 4-((4-oxo-4- (pentadecane-8-yloxy) butyl) amino) butanoate (1 equivalent) dissolved in 250 mL of dry DCM To the solution of was added 13.5 g of triphosgene (1 eq), the reaction mixture was cooled to 0 ° C., and 18.4 mL of pyridine (5 eq) was added dropwise over 10 minutes. The reaction mixture was stirred at 20 C for 4 hours.

  DCM was removed under reduced pressure and the mixture was collected with pyridine (300 mL). After cooling to 0 ° C., 32.3 g of dimethylaminoethanethiol hydrochloride (5 eq.) Were added in small portions and the resulting solution was stirred overnight at 20 ° C. under a nitrogen atmosphere.

After TLC confirmation, the reaction mass was concentrated to dryness under reduced pressure. To this residue was added 250 mL EA and 200 mL (10%) citric acid. The organic phase was separated, then the organic layer was washed once again with 10% citric acid (100 mL) and once again with 10% brine (200 mL). The obtained organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain a crude product.

  The first purification was performed on silica gel (100-200 mesh; 500 g). For gradient elution, the compound was eluted with 70% EtOAc / hexane. After concentration, the compound (22.0 g) appeared reddish. A second purification was performed using neutral alumina (400 g) with a HPLC grade solvent. The compound was eluted with 15% EtOAc / hexane and the pure fractions were concentrated under reduced pressure to give 19.0 g of a yellow liquid.

  The product (14.0 g) was diluted with 200 mL of EtOH (HPLC grade), then 7.0 g of charcoal (50% W / W) was added to the solution and stirring was continued at room temperature overnight. The resulting solution was filtered through a pad of celite and the filtrate was concentrated. Finally, dissolved in 120 mL of 50% EtOAc / hexane, filtered through a cotton plug (to remove alumina and silica particles) and concentrated under reduced pressure. The final compound (11.5 g) appeared pale yellow.

  The second batch (5.0 g) was diluted with 80 mL of EtOH (HPLC grade), then 2.5 g of charcoal (50% W / W) was added to the solution and stirring was continued at room temperature overnight. The resulting solution was filtered through a pad of celite and the filtrate was concentrated. Finally, it was dissolved in 60 mL of 50% EtOAc / hexane, filtered through a cotton plug (to remove alumina and silica particles) and concentrated under reduced pressure. The final compound (4.0 g) appeared reddish yellow. Production amount: 15.5 g; yield: 51%.

ATX-0081: ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 5.62 (m, 1), 5.51 (m, 1), 4.86 (m, 1), 4.62 ( d, J = 6.0 Hz, 2), 3.37 (brs, 4), 3.01 (t, J = 7.1 Hz, 2), 2.51 (t, J = 7.1 Hz, 2), 2.31 (m, 4), 2.26 (s, 6), 2.07 (m, 2), 1.89 (brs, 4), 1.42 to 1.57 (4), 1.16 ~ 1.40 (28), 0.82 to 0.91 (9)
Example 15: Synthesis of ATX-0085

  FIG. 15 shows the synthesis route for ATX-0085, which is described further below.

ATX-0085: Step 1

  In a 500 mL single neck RB flask was charged 30.0 g of heptanoic acid (1 eq.) Dissolved in DCM (200 mL) and then 26.7 mL of oxalyl chloride at 0 ° C. with stirring under a nitrogen atmosphere. (1.5 eq) were added slowly, followed by ImL of DMF (catalyst). The resulting reaction mixture was stirred at room temperature for 2 hours.

  In a separate 1 liter two-necked RB flask, 46.6 g of N, O-dimethylhydroxylamine hydrochloride (2 eq) in DCM (250 mL) was stirred at 0 ° C. using an addition funnel at 86.6 mL. Of trimethylamine (3 equivalents) was added. After concentration under reduced pressure to the resulting solution, the acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (100 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (250 mL).

  The organic layer was separated and the aqueous layer was washed with DCM (3x100mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 silica gel) using 10% EtOAc / hexane. 38.0 g produced; 84% yield.

ATX-0085: Step 2

  38.0 g dissolved in 250 mL ether in a solution of 62.3 g hexylmagnesium bromide (1.5 eq) in ether, placed in a 1 L two necked round bottom RB flask and stirred at 0 ° C. under nitrogen atmosphere. Of N-methoxy-N-methylheptaneamide (1 equivalent) was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7).

The reaction mass was quenched with saturated NH ₄ Cl solution (200 mL). The organic layer was separated and the aqueous layer was washed with ether (2 × 100 mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 2% EtOAc / hexane. 30.8 g produced; 71% yield.

ATX-0085: Step 3

  To a solution of 30.0 g of tridecane-7-one (1 eq) dissolved in 200 MeOH / THF (10: 1, v: v) was added 8.5 g of sodium borohydride (1.5 eq) at 0 ° C. Was added and the resulting solution was stirred at room temperature for 2 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5).

The reaction mass was quenched with a saturated NH ₄ Cl solution (80 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (200 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 70 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. 27.2 g produced; 90% yield.

ATX-0085: Step 4

  To a solution of 50.0 g of 4-aminobutanoic acid (1 equiv.) Dissolved in THF / aqueous NaOH solution (490 mL) at 0 ° C., followed by 140 mL of Boc anhydride (1.3 equiv.) Using an addition funnel. For 15 minutes. The resulting solution was stirred at room temperature for 4 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.5)} .

  The reaction mass was quenched with 5% HCl (250 mL) and then EtOAc (300 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3x150mL). The combined organic layers were concentrated under reduced pressure to give a rubbery liquid. 80.0 g produced; 81% yield. Confirmed by 1H NMR. Note: Boc-acid (Int4) with some impurities (20-30%) was identified by 1H NMR, which was reflected in the yields of Step 5 and Step 6.

ATX-0085: Step 5

A solution of 10.0 g of 4-((tert-butoxycarbonyl) amino) butanoic acid (1 equivalent) dissolved in DCM (200 mL) and cooled below 0 ° C. was charged with 12.2 g of EDC. HCl (1.3 equiv), _Et 3 N (3 eq) in 20.4 mL, and 601mg of DMAP (0.1 eq) were sequentially added at 10 minute intervals under a nitrogen atmosphere. To the resulting solution was added alcohol at the same temperature by dissolving in DCM (150 mL) using an addition funnel and stirring at room temperature under nitrogen atmosphere for 24 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.5).

The reaction mass was quenched with water (150 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x75mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (100 mL) and then EtOAc (200 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. 8.0 g of product (crude; required compounds and alcohols).

ATX-0085: Step 6

  To a solution of 8.0 g of tridecane-7-yl 4-((tert-butoxycarbonyl) amino) butanoate (1 eq.) Dissolved in 65 mL of DCM at 0 <0> C was added 15.7 mL of TFA (10 eq.). The mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours.

The progress of the reaction was monitored by TLC (10% in _{CHCl 3 MeOH; Rf: 0.3)} .

The reaction mass was concentrated under reduced pressure. The resulting crude was washed with saturated NaHCO ₃ solution (100 mL) and then extracted with EtOAc (3 × 100 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography using (60-120 mesh silica gel; 4% MeOH / CHCl ₃ and 1 mL of triethylamine) to recover the alcohol. 3.5 g yield; 25% yield in two steps; confirmed by mass.

ATX-0085: Step 7

  To 20.0 g of hexanoic acid (1 eq.) Dissolved in DCM (150 mL) was slowly added at 0 ° C. with stirring under nitrogen atmosphere 22.1 mL of oxalyl chloride (1.5 eq.) Followed by 1 mL Of DMF (catalyst) was added. The resulting reaction mixture was stirred at room temperature for 2 hours.

  In a separate flask, 31.7 g of N, O-dimethylhydroxylamine hydrochloride (2 eq) in DCM (250 mL) was stirred using an addition funnel at 0 ° C. with 71.7 mL of trimethylamine (3 eq). Was added. After concentration under reduced pressure to the resulting solution, the acid chloride was added dropwise over 20 minutes using an addition funnel by dissolving in DCM (100 mL) under a nitrogen atmosphere. The obtained reaction solution was stirred at room temperature for 3 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.5). The reaction mass was diluted with water (250 mL). The organic layer was separated and the aqueous layer was washed with DCM (2x100mL). The combined organic layers were concentrated under reduced pressure.

  The crude compound was subjected to column chromatography (60-120 silica gel) using 10% EtOAc / hexane. Production amount 21.0 g; yield 76%.

ATX-0085: Step 8

  Under a nitrogen atmosphere, at 0 ° C., a solution of 33.0 g of pentylmagnesium bromide (1.5 eq.) In ether was dissolved in 220 mL of ether with 20.0 g of N-methoxy-N-methylhexanamide (1 eq.). ) Was added and the resulting reaction mixture was stirred at room temperature for 4 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.6).

The reaction mass was quenched with saturated NH ₄ Cl solution (200 mL). The organic layer was separated and the aqueous layer was washed with ether (2 × 100 mL). The combined organic layers were dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 2% EtOAc / hexane. Production amount: 15.4 g; yield: 72%.

ATX-0085: Step 9

  To a solution of 15.0 g of undecane-6-one (1 equiv) dissolved in 100 mL of MeOH / 15 mL of THF at 0 ° C. was added 4.9 g of sodium borohydride (1.5 equiv) to give a solution. The solution was stirred at room temperature for 2 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5).

The reaction mass was quenched with a saturated NH ₄ Cl solution (80 mL). The solvent was removed under reduced pressure and the resulting crude was partitioned between EtOAc (200 mL) and water (100 mL). The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 70 mL). The combined organic layers were concentrated under reduced pressure to give a white solid. 13.9 g produced; white solid; 92% yield

ATX-0085: Step 10

To a solution of 10.0 g of 5-bromopentanoic acid (1 equivalent) dissolved in DCM (300 mL) and cooled below 0 ° C. was added 13.7 g of EDC. HCl (1.3 eq), 23.0 mL of _Et 3 N (3 eq), and DMAP in 674mg under a nitrogen atmosphere, were successively added at 10 minute intervals. To the resulting solution was added 9.5 g of alcohol (1 equivalent) at the same temperature by dissolving in DCM (150 mL) using an addition funnel and stirring at room temperature under a nitrogen atmosphere for 24 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.7).

The reaction mass was quenched with water (200 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2 × 100 mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was washed with a saturated NaHCO ₃ solution (100 mL) and then EtOAc (3 × 100 mL) was added. The organic layer was separated, concentrated under reduced pressure and carried on to the next step with the crude. The crude compound was subjected to column chromatography (60-120 mesh silica gel) using 5% EtOAc / hexane to recover SM (alcohol). Production amount 11.6 g; yield 62%.

ATX-0085: Step 11

  A solution of 4.5 g of tridecane-7-yl 4-aminobutanoate (1 eq) in ACN, 5.2 g of undecan-6-yl 5-bromopentanoate (1 eq) was added with 3.0 g of carbonic acid. Potassium (1.4 eq) was added and the resulting mixture was refluxed at 90 ° C. for 4 hours under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.45).

  The reaction mass was filtered, washed with ACN (2 × 20 mL) and the filtrate was concentrated under reduced pressure. The crude compound was subjected to column chromatography (100-200 mesh silica gel) using 20% EtOAc / hexane. SM (amine and bromo compound) was recovered. Yield, 2.2 grams of pure compound; 25% yield; and 1.1 grams of mixture. Confirmed by mass.

ATX-0085: Step 12

  To a solution of 2.2 g of undecane-6-yl 5-((4-oxo-4- (tridec-7-7-yloxy) butyl) amino) pentanoate (1 equivalent) dissolved in dry DCM was added 1.6 mL of trimethylamine ( 3 eq) and 604 mg of triphosgene (0.5 eq) were added at 0 ° C. at 5 min intervals under a nitrogen atmosphere. The resulting solution was stirred at room temperature for 1 hour under a nitrogen atmosphere. The resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere.

  Under a nitrogen atmosphere, a suspension of 684 mg of sodium hydride (7 eq) in dry THF (30 mL) was added in a stirred flask at 0 ° C. to 2.0 g of 2- (dimethylamino) ethane-1-thiol. The hydrochloride (3.5 eq) was added and stirring continued under a nitrogen atmosphere for 5 minutes. To the resulting solution, the carbonyl chloride, dissolved in THF (50 mL), was added slowly via syringe over about 10 minutes. The resulting solution was stirred overnight at room temperature under a nitrogen atmosphere.

The progress of the reaction was monitored by TLC (10% MeOH / CHCl ₃ ; Rf: 0.5; PMA carbonization).

The reaction mass was quenched with saturated NH ₄ Cl (40 mL) and then EtOAc (100 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2x40mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography. The first purification was performed using silica gel (100-200 mesh). 5.0 g of the crude compound was adsorbed on 9.0 g of silica gel and poured onto 70 g of silica gel placed in a column. Compound was eluted with 50% EtOAc / hexane. A second purification was performed using neutral alumina with HPLC grade solvents. 2.0 g of the crude compound was adsorbed on 7.0 g of neutral alumina and the resulting was poured on 40 g of neutral alumina placed in a column. Compound eluted with 25% EtOAc / hexane. 1.4 g; yield 51%. Confirmed by ¹ H NMR.

ATX-0085 ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 4.87 (m, 2), 3.36 (brs, 4), 3.02 (t, J = 7.1 Hz, 2) , 2.52 (t, J = 7.1 Hz, 2), 2.31 (m, 4), 2.27 (s, 6), 1.88 (brs, 2), 1.56-1.66. (4), 1.46 to 1.54 (8), 1.21 to 1.34 (30), 0.89 (m, 12).
Example 15: Synthesis of ATX-0134

  FIG. 16 shows the synthesis route for ATX-0134, which is described further below.

ATX-0134: Step 1

  In a 1 L single neck RB flask, a stirred solution of 50.0 g methyl 2-bromoacetate (1 eq) in 400 mL DMF was added to 90.3 g potassium carbonate (2 eq) followed by 17.5 g benzyl The amine (0.5 eq) was added at ice bath temperature under a nitrogen atmosphere, and the resulting reaction mixture was allowed to warm to room temperature and continued stirring for 36 hours.

  The progress of the reaction was monitored by TLC (20% EtOAc / hexane; Rf: 0.4; ninhydrin carbonization).

  The reaction mass was diluted with ice water (1 L) and then EtOAc (250 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (3 × 100 mL). The combined organic layer was washed again with ice water (500 mL), and the obtained organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure.

  The crude compound was subjected to column chromatography on silica gel (60-120 mesh) using 15-20% EtOAc / hexane. 35.0 g produced; 85% yield.

ATX-0134: Step 2

  10.5 g of 10% Pd / C is added to a reaction 500 mL hydrogenation flask containing 35.0 g of dimethyl 2,2 ′-(benzylazanediyl) diacetate in EtOAc and the starting material is consumed (2. H), the reaction mass was hydrogenated using a Parr shaker (60 psi).

  The progress of the reaction was monitored by TLC (5% MeOH / CHCl3; Rf: 0.5; ninhydrin carbonization).

  The reaction mass was filtered through a pad of celite and washed with EtOAc (2 × 60 mL). The combined filtrate was concentrated under reduced pressure. Production amount 30.0 g (crude product).

ATX-0134: Step 3

  To a solution of 30.0 g of dimethyl 2,2'-azanediyl diacetate (1.0 eq) in THF is added 38.7 mL of triethylamine (1.5 eq) followed by 55.5 mL of Boc anhydride (1. 3 eq.) Were added sequentially using an addition funnel at ice bath temperature. The resulting reaction solution was stirred overnight at room temperature under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (40% EtOAc / hexane; Rf: 0.5).

  The reaction mass was diluted with water (250 mL) and then EtOAc (150 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was subjected to column chromatography on silica gel (60-120 mesh) using 15% -20% EtOAc / hexane. 35.0 g produced; 96% yield.

ATX-0134: Step 4

  To a solution of 35.0 g of dimethyl 2,2 ′-((tert-butoxycarbonyl) azanediyl) diacetate (1.0 eq) in THF stirred at ice bath temperature was added 16.8 g of lithium hydroxide (75 mL of 5.3M) (3 eq) aqueous solution was added. The resulting reaction solution was stirred at room temperature for 5 hours under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (10% MeOH / CHCl3; Rf: 0.2).

  The reaction mass was quenched with 5% HCl (400 mL) and then EtOAc (200 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound was subjected to column chromatography on silica gel (60-120 mesh) using 100% EtOAc. Production amount 29.0 g; yield 93%.

ATX-0134: Step 5

  12,2 '-((tert-butoxycarbonyl) azanediyl) diacetate 233.22 0.051 1.0

  2 alcohol 228.42 0.102 2.0

  3 EDC. HCl 191.70 0.154 3.0

  4 Triethylamine 101 0.154 3.0

  5 DMAP 122.17 0.002 0.1

  6 DCM 400mL

To a solution of 12.0 g of 4,4-((tert-butoxycarbonyl) azanediyl) dibutanoic acid (1 equivalent) in 250 mL of DCM cooled to 0 ° C. to 50 ° C. (ice bath) was added 29.5 g of EDC. HCl (3 eq), 21.4 mL of _Et 3 N (3 eq), and 628mg of DMAP (0.1 eq) under a nitrogen atmosphere, were successively added at 10 minute intervals. To this resulting solution, 23.5 g of alcohol (in 150 mL of DCM) (2 eq) is added via addition funnel at the same temperature and the resulting reaction mass is warmed to room temperature and treated under nitrogen atmosphere for 24 hours. Stirred for hours.

  The progress of the reaction was monitored by TLC (10% EtOAc in hexane; Rf: 0.6; PMA charring).

The reaction mass was quenched with water (100 mL) and then the organic layer was separated. The aqueous layer was washed with DCM (2x80mL). The combined organic layers were concentrated under reduced pressure. The resulting crude was stirred with a saturated NaHCO3 solution (100 mL) for 5 minutes to remove unreacted acid, then EtOAc (2 x 80 mL) was added. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The crude compound was subjected to column chromatography on silica gel (60-120 mesh) using 2-3% EtOAc / hexane. Production amount 15.0 g; yield 44%.

ATX-0134: Step 6

  A solution of 15.0 g of di (pentadecan-8-yl) 2,2-((tert-butoxycarbonyl) azanediyl) diacetate (1 equivalent) in 120 mL of DCM was added at 0 ° C. to 50 ° C. (ice bath). 0.7 mL of TFA (10 eq) was added and the reaction mass was warmed to room temperature and stirred under a nitrogen atmosphere for 4 hours.

  The progress of the reaction was monitored by TLC (10% EtOAc / hexane; Rf: 0.5).

  The reaction mass was concentrated under reduced pressure, the resulting crude was stirred with saturated NaHCO3 solution (100 mL) for 5 min (to remove traces of TFA) and the aqueous phase was extracted with EtOAc (3 x 100 mL). The organic layer was separated and concentrated under reduced pressure. The crude compound was subjected to column chromatography on silica gel (60-120 mesh), eluting the compound with 10% EtOAc / hexane. 10.4 g produced; 82% yield.

ATX-0134: Step 7

  To a solution of 1.5 g of di (pentadecan-8-yl) 2,2'-azandiyl diacetate (1 eq) in 20 mL of dry DCM, 1.1 mL of trimethylamine (3 eq) and 401 mg of triphosgene (0. 5 equivalents) were added at 5 minute intervals under ice bath temperature and nitrogen atmosphere. The resulting solution was then warmed to room temperature and stirred for 1 hour under a nitrogen atmosphere. After completion of the starting material (confirmed by TLC), the resulting reaction mass was concentrated under reduced pressure and maintained under a nitrogen atmosphere. 1.1 g of 3- (diethylamino) propane-1-thiol (3 equiv.) Dissolved in 10 mL of THF was cooled to 0 ° C. to 50 ° C. under a nitrogen atmosphere and 129 mg of sodium hydride in dry THF (10 mL) (2 eq) suspension and stirring continued for 5 minutes. To the resulting solution, the carbamoyl chloride, dissolved in THF (30 mL), was added via an addition funnel at the same temperature over about 5 minutes. The resulting solution was warmed to room temperature and kept stirring overnight under a nitrogen atmosphere.

  The progress of the reaction was monitored by TLC (10% MeOH / CHCl3; Rf: 0.5; PMA carbonization).

  The reaction mass was quenched with saturated NH4Cl (30 mL), then EtOAc (50 mL) was added. The organic layer was separated and the aqueous layer was washed with EtOAc (2x40mL). The combined organic layers were concentrated and the resulting crude was subjected to column chromatography. The first purification was performed using neutral alumina (100 g). For gradient elution, the compound was eluted with 20% EtOAc / hexane. A second purification was performed using silica gel (100-200 mesh; 80 g) with a HPLC grade solvent. Compound was eluted with 50% EtOAc / hexane. 680 mg; yield 34%.

ATX-0134 ¹ H-NMR (PPM, 400 MHz, CDCl ₃ ): δ = 4.91 (m, 2), 4.21 (s, 2), 4.16 (s, 2), 2.95 (t , J = 7.1 Hz, 2), 2.54 (m, 6), 1.79 (m, 2), 1.46 to 1.52 (8), 1.17 to 1.36 (40), 1.03 (t, J = 7.8 Hz, 6), 0.87 (t, J = 7.1 Hz, 12).
Example 15: Synthesis of ATX-0044 and ATX-0091-ATX-0133.

ATX-0044, ATX-0085, ATX-0111, ATX-0132, ATX-0100, ATX-0117, ATX-0114, ATX-0115, ATX-0101, ATX-0106, ATX-0116, ATX-0122, ATX- 0123, ATX-0124, ATX-0126, ATX-0129, and ATX-0133 were synthesized using the method of the previous example.
Example 16: pKa value

The pK _a of _the cationic lipid LNP or micellar formulation, Jayaraman, 2012, Angew. Chem. Int. Ed. , 51: 8529-33 (incorporated herein by reference). Lipid micelles or LNPs were treated with a pH sensitive fluorescent probe, 3-12, in the presence of 0.06 mg / mL 6- (p-toluidino) -2-naphthalenesulfonic acid sodium salt (TNS) reagent (Sigma Aldrich). Dilute to 1 mM total lipids in a universal buffer with a pH range of Anionic TNS molecules fluoresce when associated with the surface of a positively charged membrane, but do not fluoresce when free in solution, allowing pKa measurements. TNS signals are measured on a spectrum plate reader. The TNS signal was plotted as a function of pH, determining the pK _a and analyzed using a non-linear (Boltzmann).

Reagents used in the assay include:
Hepes free acid, CAS: 7365-45-9 (VWR, 0511-1KG or equivalent)
-MES, HPLC grade: (Sigma 105228-100G or equivalent)
-Ammonium acetate, HPLC grade: (Sigma 90335-100mL or equivalent)
・ Sodium chloride, HPLC grade (VWR EM-MX0475-1 or equivalent)
-TNS (Sigma-Aldrich T9972-250mg or equivalent)
・ Hydrochloric acid ・ DMSO
-Calcium and magnesium free DPBS (GE, SH30028.O2 or equivalent)
· _H 2 O, HPLC grade (OmniSolv, WX0004-1 or equivalent) can be mentioned.

試薬は、０．２μｍフィルターを通して滅菌濾過される。ＵＢ溶液の調製には、５ｍＬの１Ｍ ＨＣｌを３５０ｍＬの原液に添加することが含まれる。 To prepare a stock solution of Universal Buffer Solution (UB) in a sterile polystyrene storage bottle, prepare 1 L using the table below.

Reagents are sterile filtered through a 0.2 μm filter. Preparation of the UB solution involves adding 5 mL of 1 M HCl to 350 mL of the stock solution.

  To prepare the pH ladder, use a centrifuge tube (50 mL) with 20 mL of UB buffer. (PH about 3) and different amounts of 2N NaOH. Stock solution of 1 mg / mL TNS in DMSO. Add 60 μL of TNS (1 mg / mL) to 940 μL of water to obtain a working solution concentration of 0.06 mg / mL. Five test samples (1 mL LNP sample in PBS at 1 mM total lipid) and one reference sample are analyzed per plate. Samples of different pH values are prepared in wells to which 25 μL of 0.06 mg / mL TNS has been added. Add 15 μL of test sample per well. Incubate the microplate in the dark for 15 minutes at room temperature. The TNS template can be used to format data from a plate reader. Perform a non-linear (Boltzmann) regression analysis of the sample in Origin Pro 8 or equivalent software.

  Table 1 shows the results. In general, increasing lipophilicity via an ester near the ionizable head group will lower the measured pKa while making the calculated pKa more basic. Adding lipophilicity to alcohols while reducing the length of the ester chain can have a significant effect on the measured pKa. For example, shortening the ester to butanoate has no effect when the lipophilicity of the alcohol is increased to a branched alkyl group (see ATX-0057 and ATX-0058 versus ATX-0002). The great effect is that when shortening the ester to acetate, for example, ATX-0061 / ATX-0064 (Δ = −0.90) and ATX-0063 (1-branched / 1-Z-2-nonenol ester) Δ = -0.7), as well as the acetate / bis-branched ester ATX-0062 / ATX-0065 (Δ = -3.20) (ΔcLogP + 4.0) versus ATX-0002.

Without being bound by theory, the results show that, compared to expected, increased lipophilicity (as measured by cLogP) in combination with chain shortening in reducing the measured pKa, and the lipid nanoparticles Shows the importance of increasing biological activity.
Example 17: In vivo EPO mRNA stability

The levels of mRNA in plasma were measured and compared after injection of nanoparticles containing different cationic lipids. Female Balb / c mice (6-8 weeks old) were used to assess plasma erythropoietin (epo) levels in vivo after injection of lipid-encapsulated mouse epo mRNA. All formulations were administered intravenously via tail vein injection at doses of 5 mL / kg, at doses of 0.03 and 0.1 mg / kg. Six hours after formulation injection, terminal blood collection was performed via cardiac puncture under 2% isoflurane. Blood was collected in 0.109 M citrate buffer tubes and processed by centrifugation at 5000 rpm for 10 minutes. Serum was collected and analyzed for epo mRNA levels. The results are shown in FIG. The results show a substantial improvement over ATX-0002 of ATX-0057, ATX-0081, ATX-0082, ATX-0083, ATX-0084, ATX-0085, ATX-0087, and ATX-0087.
Example 18: In Vivo Mouse Factor VII Silencing and EPO Expression

  Using a liver-directed in vivo screen of a liposome library, a series of compounds that promote high levels of siRNA-mediated gene silencing in hepatocytes (cells include liver parenchyma) were tested. Factor VII, a blood clotting factor, is a suitable target gene for assaying functional siRNA delivery to the liver. Since this factor is specifically produced in hepatocytes, gene silencing shows good delivery to the parenchyma, as opposed to delivery to cells of the reticuloendothelial system (eg, Kupffer cells). In addition, Factor VII is a secreted protein that can be easily measured in serum and does not require euthanizing animals. Silencing at the mRNA level can be readily determined by measuring protein levels. This is due to the short half-life of the protein (2-5 hours). Compositions with siRNA directed to factor VIII were formulated with lipids and a comparative sample, phosphate buffered saline (PBS). Female C57BL / 6 mice (6-8 weeks old) were used for FVII siRNA knockdown (KD) experiments.

  All formulations were administered intravenously via tail vein injection at doses of 5 mg / kg, at doses of 0.03 and 0.1 mg / kg. Forty-eight hours after formulation injection, terminal blood collection was performed via cardiac puncture under 2% isoflurane. Blood was collected in 0.109 M citrate buffer tubes and processed by centrifugation at 1200 G for 10 minutes. Plasma was collected and factor VII protein levels were analyzed by a chromogenic assay (Biophen FVII, Aniara Corporation). A standard curve was constructed using samples from PBS-injected mice, and relative factor VII expression was determined by comparing the treated group to an untreated PBS control. The results showed that ATX-0057 and ATX-0058 were substantially more effective than ATX-002 at both 0.03 and 0.1 mg / kg (FIG. 18).

  Table 1 shows the knockdown obtained from lipid nanoparticles comprising the lipids disclosed herein.

  Epo expression was measured after delivery of mRNA using lipid nanoparticles comprising the lipids described herein.

  Female Balb / c mice (6-8 weeks of age) were used to assess epo protein expression in vivo following delivery of lipid-encapsulated mouse epo mRNA. All formulations were administered intravenously via tail vein injection at doses of 5 mL / kg, at doses of 0.03 and 0.1 mg / kg. Six hours after formulation injection, peripheral blood collection was performed via cardiac puncture under 2% isoflurane. Blood was collected in 0.109 M citrate buffer tubes and processed by centrifugation at 5000 rpm for 10 minutes. Serum was collected and epo protein levels were analyzed by an ELISA assay (R & D systems). A standard curve was constructed using samples from PBS-injected mice, and relative factor VII expression was determined by comparing the treated group to an untreated PBS control. The results showed that epo mRNA was expressed in substantially higher amounts in ATX-0057 nanoparticles than in 0.1 mg / mL ATX-002 (FIG. 19).

Claims (20)

A compound of formula I,

Where:
R ₁ is a branched acyclic alkyl or alkenyl of 8, 9, 10, 11, 12, ₁₃ , 14, 16, 17, 18, 19, 20, 21, or 22 carbons;
L ₁ is a linear alkane of ₁ to 15 carbons,
R ₂ is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons, or 8, 9, 10, 11, 12, 13, 14 , 16, 17, 18, 19, 20, 21, or 22 branched acyclic alkyl or alkenyl of carbon;
L ₂ is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons;
X is O or S;
R ₃ is a linear alkane of 1, 2, ₃ , 4, 5, or 6 carbons;
A compound, wherein R ₄ and R ₅ are the same or different and are each linear or branched non-cyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons;
Or a pharmaceutically acceptable salt or solvate thereof. The compound represented by the formula ATX-0082, ATX-0085, ATX-0083, ATX-0121, ATX-0091, ATX-0102, ATX-0098, ATX-0092, ATX-0084, ATX-0095, ATX-0125, ATX The method according to claim 1, wherein the compound is selected from the group consisting of compounds of -0094, ATX-0109, ATX-0110, ATX-0118, ATX-0108, ATX-0107, ATX-0093, ATX-0097, and ATX-0096. Compound.

R ₁ is, 8,9,10,11,12,13,14,16, or having 17 carbons, the compound according to claim 1. The compound of claim 1, wherein R ₁ comprises two identical alkyl or alkenyl groups. The compound of claim 1, wherein R ₁ comprises an alkenyl group. _2. The compound according to claim 1, wherein R2 is alkenyl. _2. The compound according to claim 1, wherein R2 is a branched non-cyclic alkyl. _2. The compound according to claim 1, wherein L2 has 4, 5, 6, or 7 carbons. A compound of formula I,

Where:
R ₁ is a branched acyclic alkyl or alkenyl of 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons;
L ₁ is a linear alkane of ₁ , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons;
R ₂ is linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons, or 10, 11, 12, 13, 14, 16, 17, , 18, 19, 20, 21, or 22 branched acyclic alkyl of carbon;
L ₂ is a linear alkane of 1, ₂ , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbons;
X is O or S;
R ₃ is a linear alkane of 1, 2, ₃ , 4, 5, or 6 carbons;
R ₄ and R ₅ are the same or different and are each linear, branched or non-cyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons;
A 1 mM solution of the compound has a pKa of 5.6-7.0 as measured by fluorescence of 6- (p-toluidino) -2-naphthalene sulfone;
The compound, wherein the compound has a c-LogD value of 10-14.
Or a pharmaceutically acceptable salt or solvate thereof. The compound having the formula ATX-0111, ATX-0132, ATX-0134, ATX-0100, ATX-0117, ATX-0114, ATX-0115, ATX-0101, ATX-0106, ATX-0116, ATX-0086, ATX -0058, ATX-0081, ATX-0123, ATX-0122, ATX-0057, ATX-0088, ATX-0087, ATX-0124, ATX-0126, ATX-0129, and ATX-0123 10. The compound according to claim 9, wherein

10. The compound according to claim 9, wherein R ₁ has 12, 13, 14, 16, or 17 carbons. R ₁ comprises two identical alkyl or alkenyl group, the compound according to claim 9. R ₁ comprises an alkyl group, A compound according to claim 9. R ₂ is alkenyl compound according to claim 9. R ₂ is a branched non-cyclic alkyl, A compound according to claim 9. L ₁ and _{L 2,} independently, 1, 2, or having three carbon compound according to claim 9. L ₁ and L ₂ are both having three carbon compound according to claim 9. R ₃ is propylene or butylene, A compound according to claim 9.   10. The compound of claim 9, wherein the c-LogD value is at least 11, and the measured pKa is more basic than 6. A compound of formula I,

Where:
R ₁ is a branched acyclic alkyl of 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons;
L ₁ is a linear alkane of ₁ , 2, or 3 carbons;
R ₂ is linear alkenyl of 8, 9, 10, 11, or 12 carbons, or branched acyclic alkyl of 12, 13, 14, 16, or 17 carbons;
L ₂ is a linear alkane of 1, 2, or 3 carbons;
X is O or S;
R ₃ is a linear alkane of 2 or 3 carbons;
Compounds wherein R ₄ and R ₅ are the same or different and are each linear or branched non-cyclic alkyl of 1 or 2 carbons,
Or a pharmaceutically acceptable salt or solvate thereof.

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